Haloethisterone compounds



United States Patent 3,121,079 HALQETHISTERQNE CGMPQUNDS Arthur E.Oherster, (lanton, Ghio, Roger E. Beyler, Carbondale, 111., and Lewis H.Sarett, Princeton, NJ

2 When starting with the 19nor-17a-ethynyl-4-androstene-lZB-ol-Zione, inwhich R is hydrogen, Compound II-A (and likewise Compounds H- B and HI)are mixtures of the A and the A -androstenes, and the case of the Acompound, the hydrogen (R) shown at 0-10 assignor to Mer & Co. Inc.Rahwa NJ. a cor- 5 pamfions of Newiersey is present at C-4. Removal ofthe protecting groups in No Drawing. Filed Apr. 20, 1966, Ser. No.23,394 p 3, 118mg ce t a 1 elves only the Meme/A4- 14 Cl i ((1250-43955) androstene (IV-A), :but using approximately 70% aqueousacetic acid gives a mixture of the 3-keto-A (IV-A), This invention isconcerned generally with novel stethe 3-keto-A -(IVB) and the 3-keto-Aandrostenes, roids and processes of preparing the same. Moreparrticwhich compounds can be separated by chromatography. ularly, itrelates to novel ZI-haloethisterones, ZI-halonor- When starting with the17a-ethynyl-4-androstene=17;?!- ethisterones, and closely relatedcompounds which posol-3-one, in which R is methyl, only the A-androstene is sess usefid therapeutic properties as orally andparenterpresent at IL-A, 'II-B and HI, and only the M-acndrostene allyactive progestational agents. This invention also re- (IV-A) is formedin Step 3. lates .to pharmaceutical compositions containing these Theprocess may be schematically represented in the novel steroid compounds.following manner, starting with a 17a-ethynyl-5andro- In accordance withthe present invention, the novel 21- -3 ,17p-di 1 i whi h R i ethyl, hcomespond. haloethisterones (17a-haloethynyl-4-androstene 175-01-3- ing-19. in which R is hydmgem ones) and the 21-halonorethisterones (17uhaloethynyl- 19-nor-i75-ol-3-ones) are prepared from a 17u-ethynyl-3-oxygena-ted-androstene-l7,3-01 by first protecting the HO CECE oxygenfunctions present at the C-3 and 0-17 positions of the steroid moleculewith suitable protecting groups, and then halogenating to form the17a-haloethyny1 de- 25 R rivative. The protecting groups at 0-3 and C-17are then I removed.

This process may he schematically represented as follows, starting witha 17a-ethynyl-4-androstene175-01-3- HO one in which R methyl, or thecorresponding 19-norsteroid in which R is hydrogen:

HO\ cEcH HO ,OEOH

i l Step 1 O O=c l: O

I II-A O 0 czcn 0/ 0 050K /\I R5 1 R Step 2 Step 3 o O 0 11-18 III H095021 HO 'CECX H0\ 020x RI 7 IV-O III,

110 ozox no czox If the starting material is thel7a-ethynyl-5-an'drostene- 35,17fi-diol (I), the product (IV') which isobtained on removal of the protecting groups is the 17a-lraloethyny1--androstene-3fi,l7,3-diol. This compound may be converted into the17at-haloethyny1-4-androstene-1713-01-3- one ('21-lraloethisterone) byoxidation, for example, with aluminum isopropoxide and cyclohexanone (oracetone) in benzene. The choice of the 3-oxygenated-steroid used asstarting material based on practical considerations, availability andthe like.

In the first step of our process the oxygen functions present at the 0-3and C17 positions of the steroid molecule are protected. In a preferredembodiment of our invention a 3-keto group is blocked by forming theS-ethylenedioxy-derivative by reaction with excess ethylene glycol inbenzene, toluene or ethylene dichloride solvent in the presence of acidcatalysts such as p-ttoluenesulfonic or sulfuric acid, the waterby-product being continuously removed.

The 3-keto group may also be converted into the B-ethylenedioxyderivative by exchange dioxolanation, which involves acid-catalyzedtransfer of the ethylene glycol portion of simple2,-2-dialkyl-1,3dioxolanes, such as 2,2-dimethyl-1,3-dioxolane (acetoneethylene ket-al) or, better, 2-methy-l-2-ethyl-1,3-dioxolane (butanoneethylene 4 ketal), on reaction of the ketones either in an inertsolvent, as benzene, or simply in excess reagent.

Other cyclic ketal derivatives can be used in our process for protectinga ketone group at C-3. In general, we have found that the loweralkylenedioxy derivatives wherein the hydrocarbon group contains notmore than seven carbon atoms, such as the ethylenedioxy,trimethylenedioxy, propylenedioxy and butylenedioxy derivatives are mostsuitable. However, in place of using a lower alkyienedioxy substituentto block or protect the keto substituent, we can use other derivativesreadily hydrolyzable to keto, such as an enol ether monothioketal, or adi thioketal derivative for this purpose.

The -01 group, and the 3 3,17p3-diol groups in the steroid molecule areprotected by reaction with dihydropyran in the presence of an acidicreagent, such as ptoluenesulfonyl chloride, to form the correspondingtetrahydropyranyl ether. This reaction takes place, for example, onaddition of p-toluenesulfonyl chloride to a solution of the steroid inan excess of dihydropyran. The reaction mixture is stirred at roomtemperature for 16 to 64 hours. The product may be recovered byneutralizing the reaction mixture with dilute base, and then extractingwith ether. The ether extracts are washed with water, dried andevaporated to dryness. The crude product is dissolved in a solvent suchas petroleum ether, and chromatographed over alumina to give thecorresponding tetrahydropyranyl derivative.

In Step 2 of our invention, the steroid starting material, protected atthe 3- and the l7-positions as indicated above, is treated with asuitable halogenating agent. In the preferred embodiment of ourinvention, the steroid compound, dissolved in a tertiary alcohol, isfirst treated with a potassium alcoholate of that tertiary alcohol, toform the 21-potassium derivative of the steroid compound, and the lattercompound is then halogenated.

iTo form the 2l-chloro-derivative, the steroid is dissolved in t-butylalcohol and treated with potassium-tbutoxide and then witht-butyl-hypochlorite.

To form the Zl-bromo-derivative, the steroid is dissolved in t-butylalcohol and treated with potassium t-butoxide and then withN-bromosuccinimide. The choice of the tertiary alcohol reagents isdetermined by practical considerations, such as availability, solubilityand the like.

The 21-halo-steroid compounds are conveniently recovered by adding waterto the reaction mixture, and extracting with ether. The ether extractsare then washed with water, dried, and evaporated to dryness in vacuo..The residual material thus obtained is separated by chromatography onalumina to aiford the 2l-halo-steroid.

in Step 3 of our process, the protecting groups at C3 and C-17 areremoved, preferably by treatment with acid. Removal of the protectinggroups using concentrated HCl gives the corresponding 3-keto-A-androstene. To carry out this reaction, a solution of the steroid inmethanol is stirred with concentrated HCl for approximately one hour atroom temperature. The methanol may then be removed under reducedpressure. The steroid is conveniently recovered by ether extraction. Theether extract is dried, evaporated to dryness and the residual materialcrystallized from a suitable solvent.

When starting with the l9-nor-17ot-ethynyl-4-androstene-17B-ol-3-one, inwhich R is hydrogen, removal of V aluminum isopropoxide andcyclohexanone in benzene. This oxidation is carried out by dissolvingthe steroid in a solvent such as a mixture of cyclohexanone and benzeneand reacting in an inert atmosphere with a solution or" aluminumisopropoxide in benzene at 8090 C. for 2-16 hours. To recover theproduct, the solution is cooled, a few drops of water are added, and theresulting alumi num hydroxide is filtered off. The filtrate is concentrated on the stream bath under reduced pressure, and the residualmaterial is crystallized from a suitable solvent.

In accordance with the present invention, the novel 21- haloethisteronesare prepared from the l7a-ethynyLS-androstene-3fl,17B-diol by firstreacting the latter compound with dihydropyran in the presence ofp-toluenesulfonyl chloride to give the 17cz-ethynyI-5-androstene-35,17fl-diolbis-tetrahydropyranyl ether.

The 17a-ethymyl-5-androstene-3,8,l7fi-diol-bis-tetrahydropyranyl etheris chlorinated by dissolving in t-butyi alcohol and treating with asolution of potassium t-butoxide and then with t-butyl 'hypochlorite togive the 17 ozchioroethynyl-5-androstene-3B,17,8-diol bistetrahydropyranyl ether. The latter compound is then hydrolyzed withconcentrated HCl to give 17a-chloroethynyl-5-androstene-3fi,17fi-diol,Which is then oxidized With aluminum isopropoxide and cyolohexanone inbenzene to give the 17a-chloroethynyl-4-androstene-175-01-3-one (21-chloroethisterone) The 17stethynyl-5-androstene-3fi,17fl-diol-bis-tetrahydropyranyl ether isbrominated by dissolving the steroid in t-butyl alcohol and treatingwith a solution of potassium t-butoxide and then with N-bromosuccinimideto give thel7a-bromoethynyl-5-androstene-3B,17fl-diol-bistetrahydropyranyl ether.The latter compound is then hydrolyzed with concentrated HCl to givel7a-bromoethynyl-5-androstene-3t3,175-diol, which is then oxidized withaluminum isopropoxide and cyclohexanone in benzene to give the17ot-bromoethynylt-androstene-175-01- 3-one (21-'bromo-ethisterone).

The novel 21-halonorethisterones are prepared from norethisterone(17a-ethynyl-19-nor-4-androstene-17fi-ol- 3-one) by reaction first withethylene glycol in the presence of p-toluenesulfonic acid to give amixture of the A and the A-17a-ethynyl-3-ethylenedioxy-l9-norandrostene-l7fi-ol, and then withdihydropyran to give the corresponding tetrahydropyranyl ether.

The mixture of the A and the A -l7a-ethynyl-3-ethylenedioxy-l9-nor-androstene-1713-01 tetrahydropyranyl ether ischlorinated by dissolving the steroid in t-butyl alcohol and treatingwith a solution of potassium t-butoxide and then witht-butylhypochlorite to give a mixture of the A and the A-17a-chloroethynyl-19-nor-androstene-17I3-ol-3-one tetrahydropyranylether, which mixture is then hydrolyzed With acid. Hydrolysis of themixture with concentrated HCl gives the l7a-chloroethynyl- 19 nor 4androstene 17,8 ol 3 one (21 chloronorethisterone). Hydrolysis of themixture with 70% aqueous acetic acid gives a mixture of the A the A500)and the A -17ct-chloroethynyl-l9-nor-androstene-l7B-ol- 3-ones, whichmixture is separated by chromatography.

The mixture of the A and the A -17cz-ethynyl-3- ethylenedioxy 19 norandrostene 175 ol tetrahydropyranyl ether is brominated by dissolvingthe steroid in t-butyl alcohol and treating with a solution of potassiumt-butoxide and then with N-bromosuccinimide to give a mixture of the Aand the A -17a-bromoethynyl-19- nor androstene 17 8 ol 3 onetetrahydropyranyl ether. The latter compound is then hydrolyzed Withacid. Hydrolysis of the mixture with concentrated HCl gives the17a-bromoethyny1-19-nor-4-androstene-175-01-3- one(21-bromonorethisterone). Hydrolysis of the mixture with 70% aqueousacetic acid gives a mixture of the A the A and the A-17a-bromoethynyl-19-nor-androstene-17fl-ol-3-ones, which mixture isseparated by chromatography.

In addition to the above described 17a-haloethynyl-4 5 androstene-173-ol-3-ones, this invention contemplates the preparation of certainderivatives thereof, and in particular the novel steroid compounds whichmay be chemically represented as follows:

wherein the 1,2-positions and the 6,7-positions of the steroid moleculemay be double bonded or saturated, and wherein R is hydrogen, a methylgroup, R is a lower alkyl or alkanoyl group, X is CECCI, CECBI', CH=CHCLCH=CHBr, CH CH Br or CH CH CI, and Y is methyl, chloro or fiuoro.

This invention also contemplates the preparation of the l7fi-lower alkylesters and ethers of the 17u-haloethynyl-19-nor-5(1G)-androstene-17,8-01-3-011e, as well as the products whichare formed on hydrogenating the 17mhaioethynyl group of the aforesaidcompounds to the CH=CHX, and to the 17aCH CH X groups, where Xrepresents chlorine or bromine.

The 17a-haloethyny1 group of the 21-haloethisterones and theZl-halonorethisterones (Vi) is reduced to the corresponding chlorovinylderivative (VI) on hydrogenation with a Lindlar catalyst, and to thecorresponding chloroethyl derivative (VII) on hydrogenation using aheavy metal catalyst such as PtO n'o on-z-cnx VII dized with anoxidizing reagent prepared from chromium trioxide and sulfuric acid togive 6B-methy1-17a-haloethynyl-androstane-Sa,17B-diol-3-one, which ontreatment with aqueous sodium hydroxide gives the6a-methyl-l7ahaloethynyl-4-androstene-175-ol-3-one.

The 6u-methyl-17a-haloethynyll-androstene-17 6-01-3- one isdehydrogenated at C-l by means of selenium dioxide, or alternately bymicrobiological methods, to give the 6a-methyl-l7a-haloethynyl- 1,-4androstadiene-Ufi-ol- 3-one.

The 6a-methyll7a-haloethynyl-4-androstene-l7(3-01-3- one is reacted withchloranil to give 6-.ethyl-17a-haloethynyl-4,6-androstadiene-17,8-01-3-one, which compoundis dehydrogenated at C-1, by means of selenium dioxide, or alternatelyby microbiological methods, to give 6- methyl 17a haloethynyl 1,4,6androstatriene 17B- ol3-one.

The l7fi-lower alkyl ethers of thel7B-hydroxy-17uhaloet-hynyl-3-ketosteriods are prepared by the reactionof the corresponding l7B-hydroxy-steroid with a lower alkyl halide andsilver oxide in a solvent such as dimethylformamide. The lower alkylhalides which may be used for this purpose includes methyl iodide, ethyliodide, npropyl iodide, n-butyl iodide and the like.

The l7/3-lower alkanoyl ethers of thel7fi-hydroxy-l7ahaloethynyl-3-keto-steroids are prepared by the reactionof the corresponding 17p-hydroxy-steroid with a lower alkanoic acidanhydride in the presence of an organic base such as pyridine. The loweracid anhydrides which may be used for this purpose include aceticanhydride, propionic anhydride, butyric anhydride, and the like.

The 17a-haloethynyl-l,4-androstadiene-17B-ol-3-one acetate is preparedby reacting 17u-hal0ethynyl-4-androstane-l7fi-ol-3-one with aceticanhydride in pyridine to give the corresponding acetate, and thenconverting the latter compound into the 17a-haloethynyl-l,4-androstadiene-l75-ol-3-one acetate by means of selenium dioxide, or alternatelyby microbiological methods.

The 17cc haloethynyl-1,4-androstadiene-17B-methoxy- 3-one is prepared byreacting l7a-haloethynyl-4-androstene-1713-ol-3-one with methyl iodideand silver oxide, using dimethylformamide as solvent, to give the1705-haethynyl 4 androstene-17,8-meth0xy-3-one and then converting thelatter compound into the l7a-haloethynyl-1,4- androstadiene17,8-methoxy-3-one by means of selenium dioxide, or alternately bymicrobiological methods.

The 6a-chloro-l7tz-haloethynyl-l,4-androstadiene-l7,8- ol-3-one acetateis prepared by reacting 17u-haloethynyl- 4- androstene-l7p3-ol-3-onewith acetic anhydride and ptoluenesulfonic acid to give the17u-haloethynyl-3,5-androstadiene-3,17/3-diol-diacetate. This lattercompound is reacted with N-chlorosnccinimide to give the 6ot-chloro-17a-haloethynyl-4-androstene-175-ol-3-one acetate, which is convertedinto the 6a-chloro-l7a-haloethynyl-l,4-androstadiene-17fi-ol-3-oneacetate by reaction with selenium dioxide, or alternately bymicrobiological methods.

The 6u-chlorol7u-haloethynyl-1,4-androstadiene-17 3- methoxy 3-one isprepared by reacting 17u-haloethynyl- 4-androstene-17,8-ol-3-one withmethyl iodide and silver oxide, using dimethylformamide as solvent, togive the 17m haloethynyl 4- androstene 17 3 methoxy 3 one. The lattercompound is then converted into the 17mhaloethynyl3,5-androstadiene-17B-methoxy-3-ol acetate, by reaction with acetic acidand p-toluenesulfonic acid. The acetate so formed is treated withN-chlorosuccinimide to give the 60:chloro-l7a-haloethynyll-androstene-17(3- methoxy-3-one, which compoundis dehydrogenated by selenium dioxide, or alternately by microbiologicalmethods, to the6a-chloro-17u-haloethynyl-1,4-androstadienel7fl-methoxy-3-one.

The 6 chloro 17ct-haloethynyl-4,6-androstadiene-175- ol-3-one acetate isprepared by reaction of 17et-haloethynyl 4-androstene-l7fi-ol-3-one withchloranil to give the 170: -haloethynyl-4,6-androstadiene-l7fl-ol-3-one(A ZI-haloethisterone), which is converted into the17a-haloethynyl-4-androstene-17B-ol-3-one-6,7-oxide on reaction withperbenzoic acid. The latter compound is reacted with HCl in chloroformsolution to give the6-Cil1010-17ahaloethynyl-4,6-androstadiene-17B-ol-3-one which forms theacetate on treatment with acetic anhydride in pyridine, and thecorresponding 17,8-methoxy-derivative on reaction with methyl iodide andsilver oxide. The6-ChlO1'O-17ahaloethynyl-4,6-androstadiene-17B-ol-3-one acetate isdehydrogenated to the6-chlorodh-haloethynyl-l,4,6-androstatriene-17B-ol-3-one acetate onreaction with selenium dioxide, or alternately by microbiologicalmethods.

The 606 fluoro-17a-haloethynyl-4-androstene-175-01-3- one acetate isprepared from the 17a-haloethynyl-3,5-androstadiene- 3,17fl dioldiacetate by reaction with perchloryl fluoride followed by acidtreatment. The 60:- iluoro l7u-haloethynyl-4-androstene-17,8-01-3-oneacetate is dehydrogenated at C-1 by means of selenium dioxide, oralternately by microbiological methods, to give 60:- fluoro 17ahaloethynyl 1,4 androstadiene 17/8 ol 3-one acetate.

The Get fluoro-17a-haloethynyl-4-androstene-175-01-3- one acetate isreacted with chloranil to give6-fluoro-17ahaloethynyl-4,6-androstadiene-l7B-ol-3-one acetate, and thelatter compound is dehydrogenated at 0-1 by means of selenium dioxide,or alternately by microbiological methods, to give6-tluoro-l7a-haloethynyl-1,4,6-androstatriene-17fi-ol-3-one acetate.

The 17-haloethynyl-3,S-androstadiene-17,3-methoxy-3flol acetate isreacted with perchloryl fluoride followed by acid treatment to give6tx-fiuoro-l7a-haloethynyl-4-androstene-17fi methoxy-3-one, whichcompound may be dehydrogenated at C-1, by means of selenium dioxide, oralternately by microbiological methods, to give 6OL-fillOI'O-17a-haloethynyl-1,4-androstadiene-175-methoxy-3-one.

The 6zx-fiuoro-17a-haloethynyl-4-androstene-17/3-meth- V oxy-3-one isconverted into 6-fluoro-17ot-haloethynyl-4,6-androstadiene-17,8-methoxy-3-onc by the use of. chloranil. The6-fiuoro-l7a-haloethynyl-4,fi-androstadiene-17fi-methoxy-S-one isdehydrogenated at C-1 by means of selenium dioxide, or alternately bymicrobiological methods, to yield 6 fluorol7a-haloethynyl-1,4,6-androstatriene- 17,8-methoxy-3-one.

The 17a -haloethynyli-androstene--01-3-one is reduced to 21 halo4,20-pregnadiene-l7fi-ol-3-one by hydrogenation in ethyl acetate, usinga Lindlar catalyst, and to 21 halo-4-pregnene-l7,8-ol-3-one byhydrogenation in ethyl alcohol using a platinum oxide catalyst.

The 6a-methyl-derivatives of the 21-halonorethisterones are prepared byfirst converting the 21-halonorethisterones (17a haloethynyl 19nor-4-androstene-17fi-ol-3- one) into the 17dhaloethynyl-l9-nor-5-androstene-3/3, 17B diol-17,8-acetate by reactingthe 17u-hfl10fithYIlYl-19- nor 4-androstene-l7i8-ol-3-one with aceticanhydride and p-toluenesulfonic acid to give the l7a-haloethynyl-19-nor-3,5 androstadiene-3,l7fi-diol-diacetate and then reacting the lattercompound with sodium borohydride. Oxidation of17a-haloethynyl-l9-nor-5-androstene-3p,l7 8-dioll7fi-acetate withmonoperphthalic acid yields 17u-halo ethynyl 19 nor androstane 35,175diol 5,6 oxide 17/3 acetate. The latter compound is reacted with methyliodide and silver oxide. The6a-methyl-l7a-haloethynyl-l9-nor-androstane-3fl,5a,17 3-triol, which onoxidation with chromic oxide in acetone gives 6fl-methyl 17a haloethynyll9 nor-androstane 5a,17 3 diol 3-one. The latter compound is treatedwith sodium hydroxide to form 6amethyl-l7a-haloethynyl-19-nor-4-androstene-17fiol-3-one.

The 6a methyl 17 e-haloethynyl-19-nor-4-androstene- 17/3 ol-3-one isconverted to 6ot-methyl-17a-haloethynyl-19-nor-4-androstene-17,6-methoxy-3-one by reaction with methyl iodideand silver oxide. The6u-methyl-17c4-haloethynyl-4-androstene-17fi-ol-3-one is treated withacetic anhydride in pyridine to give 60: methyl-l7-haloethynyl-19-nor-4-androstene-l7fl-ol-3-one acetate.

The 60: chloro l7a-haloethynyl-l9-nor-4-androstene- 17fi-ol-3-oneacetate is prepared from the 17a-haloethyny1-19-nor-3,5-androstadiene-3,17B-diol diacetate by reacting withN-chlorosuccinimide.

The 6a-fluoro-17a-haloethynyl 19 nor 4-androstene- 17B-ol-3-one acetateis prepared from the 17a-haloethynyl-19-nor-3,5-androstadiene-3,17fi-diol diacetate by reacting withperchloryl fiuoride followed by acid treatment.

The 6e-chloro-17a-haloethynyl-19-nor 4 androstene- 17,8-methoxy-3-one isprepared from the 17a-haloethynyl 19-nor-4-androstene-17,8-ol-3-one byreacting first with s lver oxide and methyl iodide to form the17cz-l1fll0 ethynyl 19 nor 4-androstene-17,8-rnethoxy-3-one. The lattercompound is then reacted with acetic anhydride and p-toluenesulfonicacid to give the 17u-haloethynyl-19-nor-3,S-androstadiene-l7fi=nethoxy-3-ol acetate, which is convertedinto the Got-chloro-17a-haloethynyl-l9-nor- 4-androstene 17Bmethoxy-3-one on treatment with N- chlorosuccinimide.

The 6a fluoro-17a-1aloethynyl-19 nor 4 androstene- 17,13-methoxy-3-oneis obtained by reacting the 17 z-haloethynyl 19 nor 3,5 androstadiene17B-methoxy-3-ol acetate with perchloryl fluoride followed by acidtreatment.

The l7a-haloethynyl-l9-nor'4-androstene-17 3-ol-3-one is reduced to2l-halo-19-nor 4,20 regnadiene-17fi-ol-3- one by hydrogenation in ethylacetate, using a Lindlar catalyst and to2l-halo-19-nor-4-pre-gnene17B-ol-3-one by hydrogenation in ethylalcohol, using a platinum oxide catflyst.

The 17a-haloemynyl-19-nor-5(10) androstene-17fi-ol- 3-one forms theacetate on treatment With acetic anhydride in pyridine, Ed thecorresponding 17,8-methoxyderivative on reaction with methyl iodide andsilver oxide. The 17a-haloethynyl-l9-nor-5(l0) androstene-17 3-ol-3- oneis reduced to the corresponding chlorovinyl derivative (the21-halo-19-nor-5 (10),20-pregnadiene-l7 3-ol-3- one) on hydrogenationwith a Lindlar catalyst, and to the corresponding chloroethyl derivative(the 21-halo-19- nor pregnene 171i ol-3-one) on hydrogenation using aheavy metal catalyst such as PtO A further embodiment of our inventioncomprises novel pharmaceutical compositions containing these21-haloethisterones and 21-halonorethisterones.

Various changes and modifications may be made in the present invention,certain preferred embodiments of which are herein disclosed, Withoutdeparting from the scope thereof; to the extent that these changes andmodifications are within the scope or" the appended claims, they are tobe considered a part of this invention.

Example 1 Twenty mg. of p-toluenesulfonyl chloride is added to 400 mg.of 17a-ethynyl-5-androstene3fi,17fi-diol in 20 ml. of dihydropyran. Theresulting mixture is allowed to stand at room temperature overnight. A2.5 N NaOH solution is added until the mixture is slightly alkaline.Water is then added and the aqueous phase extracted With 4 portions ofether, each containing approximately 50 ml. The combined ether layersare washed with Water, dried over Na SO and evaporated under reducedpressure to give about 725 mg. of a non-crystalline product. The productdissolved in petroleum ether is chromatographed on 60 g. of neutralalumina and the chromatogram eluted with a 7:3 mixture of petroleumether2ether to give 400 mg. of crystalline product, the17a-ethynyl-5-androstene-3/3,17,8-diol-bis-tetrahydropyranyl ether,

A solution of about 4 grams of 17a-ethynyl-5-androstene-3,8,17,8-diolbis-tetrahydropyranyl ether in 75 ml. of t-butyl alcohol is prepared.About 1.1 equivalents of a 1.0 molar potassium =t-butoxide is added andthe resulting mixture refluxed for one hour, with stirring, Enid thencooled. About 1.84 ml. of t-butyl hypochlorite is then added in oneportion and the reaction mixture is left stirring at room temperatureovernight. About ml. of Water is added and the resulting aqueous mixtureis extracted with four portions of ether, each containing approximately200 ml. The combined layers are washed with water, dried over sodiumsulfate, filtered and evaporated to dryness in vacuo. The residualmaterial is dis solved in petroleum ether and chromatographed on g. ofalumina. Elution with petroleum ether gives about 3.10 grams (a 70%yield) of crystals of Nix-chloroethynyl-S-androstene 313,176diol-bis-tetrahydr0pyranyl ether. The crude product shows infrared peaksat 4.4;; and 2.9a.

A solution of about 3 g. of the 17u-chloroethynyl-5-androstene-3/3,17/3-diol-bis-tetrahydropyranyl ether in ml. of methanolis prepared. To this solution is added 2.5 ml. of concentratedhydrochloric acid and the reaction mixture is stirred for about 1 hourat room temperature. The methanol is then removed by evaporation underreduced pressure until the product crystallizes. Approximately 100 ml.of water is then added and the resulting product is extracted with fourportions of ether, each containing about 200 ml. The combined etherextract is Washed with water, dried over sodium sulfate and evaporatedto a crystalline residue. The residual crystalline material isrecrystallized several times from ether to give about 1.58 g. of17a-chloroethynyl-5-androstene-3B, 17fl-diol which has the followingproperties: MP. C.

Analysis.Calculated for C H O Cl: C, 72.30; H, 8.38; Cl, 10.16. Found:C, 71.64; H, 8.63; Cl, 10.48.

One hundred mg. of 17u-chloroethynyl-S-androstene- 3,9,17,6-diol isdissolved in 1.0 ml. of cyclohexanone and 10 ml. of benzene in a flaskfitted With a magnetic stirrer and a refiux condenser. About 5 ml. ofthe benzene is distilled and a stream of dry nitrogen is passed throughthe system and maintained throughout the reaction time. Then 0.5 ml. ofa 10% solution of aluminum isopropoxide in benzene is added and thereaction mixture is maintained at refiux temperature for 4 hours. Thesolution is cooled, 5 drops of water are added and the resultantaluminum hydroxide is filtered off. The filtrate is taken to drynessunder reduced pressure. The material is dissolved in ether, filtered,and the filtrate concentrated to give about 37 mg. of crude17a-chloroethynyl-4-androstene-17B-ol-3-one, M.P. 178-183 C.Recrystallization from ether gives about 25 mg. of the purified product,M.P. 182184 C. Chromatography of all mother liquors on 3 g. of aluminaand elution of the chromatogram with ether gives an additional 20 mg. ofproduct, M.P. 181- 184 C. Total yield 45 mg. The product has thefollowing properties:

U.V. A252 241 m 6 15,000. LR. kfigl? 2.8, 4.43, 6.0, 6.18;;

Analysis.Calculated for C H O Cl: C, 72.73; H, 7.85; Cl, 10.22. Found:C, 73.41; H, 7.93; Cl, 10.81.

Example 2 To a solution of 482 mg. of 17a-ethynyl-5-androstene-3B,17fi-diol-bis-tetrahydropyranyl ether in 10 ml. of tertiary-butylalcohol, is added about 1.1 equivalents of a 1.0 molar potassiumt-butoxide. The resulting mixture is refluxed for one hour, withstirring, and then cooled. 196 mg. of N-bromosuccinirnide is then addedand the reaction mixture is stirred at room temperature for about 18hours. The entire reaction mixture is dissolved in Water and thenextracted with 3 portions of ether, each containing approximately 50 ml.The combined ether extracts are washed with three portions of asaturated solu-' tion of NaHCO each portion containing approximately 251111., then with 3 portions of Water, each containing about 25 ml. Theether layer is dried over sodium sul fate, filtered and evaporated todryness. The oily residue is filtered through 20 g. of aluminum oxide togive 407 mg. of oily material which is dissolved in petroleum ether andchromatographed on 30 g. of acetone activated alkaline alumina. Elutionwith a 9:1 mixture of petroleum ether and ether yields 87 tug. of17a-bromoethynyl-5- androstene-3fi,17B-diol-bis-tetrahydropyranyl ether.An infrared spectrum of this material shows Amax 4.5a.

To a solution of 17a-bromoethynyl-S-androstene-3B,17fi-diol-bis-tetrahydropyranyl ether in 40 ml. of methanol is added 0.8ml. of concentrated HCl, and the reaction mixture is stirred for onehour at room temperature. The methanol is then removed under reducedpressure. Water is added and the resulting solution is extracted with 3portions of ether, each portion containing approximately 75 ml. Thecombined ether extracts are washed three times with approximately 50 ml.of water, dried over sodium sulfate, filtered and evaporated to dryness.The residual material is crystallized to give 230 mg. of17a-bromoethynyl-5-androstene-3,B,17fi-diol which has the followingproperties: M.P. 214215 C.

LR. mg 2.7, 2.89, 4.55;;

Analysis.(Caleulated for C21H2902B1'): C, 64.10; H, 7.43; Br, 20.32.Found: C, 62.40; H, 7.65; Br, 20.50.

17a-bromoethynyl-5-androstene-3 3,17 8-diol (195 mg.) is dissolved in1.95 ml. of cyclohexanone and 20 ml. of benzene, using a flask fittedwith a magnetic stirrer and a reflux condenser. After 3 ml. of benzeneis distilled, a stream of dry nitrogen is passed through the system, andmaintained throughout the entire reaction time. After cooling to roomtemperature, there is added 0.98 ml. of a 10% solution of aluminumisopropoxide in benzene, and the reaction mixture is refluxed for 3hours and cooled to room temperature. Ten drops of water are added andthe reaction mixture is filtered. The filtrate is taken to dryness. Theresidue is chromatographed on 15 g. of acetone activated acid-washedalumina and 85 mg. of product is eluted with a mixture of seven partsether to three parts petroleum ether. The 17a-bromoethynyl-4-androstene-17/3-ol-3-one so obtained has the following properties:

U.V. mg 240 m,., 6 15,700, LR. x535 2.0, 4.51, 6.0, 6.2,.

Analysis.(Calculated for C H O Br): C, 64.45; H, 6.95; Br, 20.42. Found:C, 65.16; H, 7.30; Br, 21.44.

Example 3 To a solution of one gram of 17a-ethynyl-19-nor-4-androstene-17fi-ol-3-one dissolved in 75 ml. of benzene is added 7.5 ml.of ethylene glycol, together with 50 mg. of p-toluenesulfonic acid. Thereaction mixture is heated at reflux with a Water separator for 20hours. The reaction mixture is cooled, and about ml. of bicarbonatesolution is added. The reaction mixture is extracted with 3 portions ofether, each portion containing about 100 ml. The combined extracts arewashed with Water, dried over sodium sulfate and evaporated to drynessto give a mixture of the A and A -17u-ethynyl-3-ethy1enedioxy-19-nor-androstene-17fi-ols.

To a solution of the total crude material from the above reaction in 10ml. of dihydropyran which has been distilled from sodium metal, is addedwith stirring, 120 mg. of p-toluenesulfonyl chloride, and the stirringis continued at room temperature for 64 hours. Saturated sodiumbicarbonate solution (10 ml.) is then added to the reaction mixture. Themixture is then extracted with 3 portions of ether, each portioncontaining about 50 ml. The extracts are combined, washed with water anddried over sodium sulfate, and evaporated to dryness in vacuo. The crudeoil (about 3.5 g.) is dissolved in petroleum ether and chromatographedon 100 g. of acetone activated alkaline alumina. The c-hromatogram iseluted with mixtures of ether and petroleum ether to give 1.043 g. of amixture of the A and A -17u-ethynyl-3-ethylenedioxy-19-nor-androstene-17,8-ol-tetrahydropyranyl ethers. The infraredspectrum shows bands at 2.9a and 9.0 4.

To a stirred solution of 2.130 g. of the mixture of the A and A-17u-ethynyl-3-ethylenedioxy-19-nor-andro- 12stene-17B-ol-tetrahydropyranyl ethers, dissolved in 50 ml. of dryt-butanol is added 5.5 ml. of 1M potassium-t-butoxide. The reactionmixture is heated at reflux for ninety minutes and then cooled to roomtemperature. Then 0.95 ml. of t-butyl hypochlorite is added. Thereaction mixture is left to stir at room temperature for about 16 hours.About ml. of water is added to the reaction mixture and the aqueoussolution is extracted with three portions of ether, each portioncontaining about 200 ml. The extracts are combined, washed with water,dried over sodium sulfate, filtered and evaporated to dryness underreduced pressure. About 2.2 g. of crude product is obtained. This ischromatographed on 100 g. of alkaline alumina. The material dissolved inpetroleum ether to which a small amount of benzene has been added ischromatographed on alumina and eluted with ether-petroleum ethermixtures. 1.9 g. of a semi-crystalline product, the 17a chloroethynyl 3ethylenedioxy-19-nor-5 [and 5(l0)]-androstene-17B-ol-tetrahydropyranylether, is obtained. Infrared absorption shows bands at 4.45 t and strongabsorption in the 910n region.

To a stirred solution of 1.9 grams of '17a-chloroethynyl- 3ethylenedioxy-19-nor-5[and 5(10)]-androstene-17fi-oltetrahydropyranyldissolved in 41 ml. of methanol is added 3 ml. of concentratedhydrochloric acid and 2.1 ml. of water. The reaction mixture is stirredat room temperature for 2% hours. The methanol is then removed underreduced pressure. The residue is dissolved in about 200 ml. of ether andwashed with water until the washings are neutral. The ether solution isdried over sodium sulfate and evaporated to dryness. The crystallineresidue, recrystallized from a mixture of ether and methylene chloride,gives 1.2 g. of 17a-chloroet-hynyl-19- nor 4 androstene--ol-3-one, M.P.194-200 C. A sample recrystallized for analysis from ethyl acetate has amelting point of 198-201 C. and shows the following properties:

U.V.: A252 240 m 6 15,000, LR; A151? 6.05, 6.2 4

Example 4 3.2 ml. of potassium t-butoxide is added to 849 mg.

of 17a-ethynyl 3 ethylenedioxy-19-nor-5[and 5(10')]- androstene-l73-ol-tetrahydropyranyl ether, prepared as in Example 3, in 28 ml. oft-buty-l-alcohol. This mixture is refluxed for 75 minutes and thencooled to room temperature. 1.136 g. of N-bromosuccinimide is added andthe reaction mixture is then stirred at room temperature for about 17hours. The reaction mixture is added to about 60 ml. of water andextracted with 3 portions of ether, each portion containing about 75 ml.The ether extracts are combined, Washed with 3, 50 ml. portions ofsaturated sodium bisulfite solution, and then with water until thewashings are neutral. The ether solution is dried over sodium sulfate,filtered and evaporated to dryness to yield the crude17a-bromoethynyl-3-ethylenedioxy- 19-nor-5 [and 5 (10)]-androstene-l7/3ol tetrahydropyranyl ether which may be used in the next step withoutpurification.

The crude steroid is dissolved in 25 ml. of methanol and 0.2 ml. ofconcentrated hydrochloric acid. The mixture is stirred at roomtemperature for 2 /2 hours. The

methanol is then removed under reduced pressure, water is added, and thereaction mixture is extracted with 3, 75 ml. portions of ether. Theether layer is Washed with water, 'ried over sodium sulfate, filteredand evaporated to dryness. This gives 737 mg. of crude oil which ischromatographed on 44 g. of acetone activated acid- Washed alumina.Elution with a mixture of ether and petroleum ether gives a crystallinematerial which is recrystallized from ether to yield about 75 mg, MP.180- 182 of 17u-bromoethynyl-l9-nor-4-androstene-l7fl-ol-3- one whichhas the following properties:

U.V.: P 239 m Emol, 16,300, I.R.: k513i? 2.85, 4.55,

max.

6.1 and 6.2

Ten grams of l7a-chloroet ynyl-S-androstene-SB,17,6- dioi in 150 ml. oftetrahydrofuran is treated with 100 ml. of 0.9 m. monoperphthalic acidin ethyl acetate and allowed to stand overnight at room temperature. Thereaction mixture is diluted with ethyl acetate and washed sequentiallwith aqueous sodium bicarbonate, sodium bisulfite, and sodiumbicarbonate solution. The organic layer is dried and concentrated invacuo. The product is fractionally crystallized from aqueous ethanol toyield the l7a-chloroethynyl-androstane-3,8,17,6-diol-5,6u-oxide.

Five grams of 17a chloroethynyl-androstane-3B,175- diol-5,6u-oxide inbenzene is added to a reagent prepared from 3.4 g. of magnesium, 10 ml.of methyl iodide and 45 ml. of ether. The mixture is stirred and 70 ml.of solvent is removed by distillation. After refluxing for 5 hours, themixture is cooled, acidified with dilute hydrochloric acid and theorganic layer washed to neutrality. The organic phase is dried andconcentrated in vacuo. Crystallization from aqueous ethanol gives the6,9methyl- 17cz-chloroethynyl-androstane-3 [3,5 ca, 17 ,G-triol.

le oxidizing reagent is prepared by diluting 2.7 g. of CrO and 2.3 ml.of concentrated sulfuric acid to ml. with Water. A solution of 1.90 g.of 6,8-methyl 17a chloroethynyl-androstane-3fi,5a,17,8-triol in 300 ml.of acetone is cooled to 0 and treated with 1.85 ml. of the oxidizingreagent. The solution is diluted with ice water and extracted withether. The ether extract is washed sequentially with water, and aqueoussodium bicarbonate andthen dried and concentrated in vacuo, to giveGil-methyl-17a-chloroethynyl-andrQStane-Se,l7fi-diol- 3-one. The crudeproduct is dissolved in 150 ml. of methanol, 75 ml. of 1 N sodiumhydroxide is added and the mixture is stirred at room temperature undernitrogen for 24 hours. The mixture is concentrated to half volume undervacuo at 25 C. and then poured into ice water. The product is extractedwith ether. The other layer is washed with water, dried andconcentrated. The concentrate is chromatographed on acid-Washed aluminaand the product eluted with ether-petroleum ether mixtures to yield6e-methyl-l7a-chloroethynyl-4-androstene- 17,8-01-3-one.

In accordance with the above procedures, but starting with the17a-bromoethynyl- -androstene-3B,17B-diol in place of the6a-methyl-17cr-chloroethynyl-4-androstene- 17 3-ol-3one, there isobtained the 6a-methyl-17a-bromoethynyl-4-androstene-17B-ol-3-one.

Example 6 To mg. of6a-methyl-17a-chloroethynyl-4-androstene-l7fi-ol-3-one in 5 ml. oft-butanol and 0.1 ml. of acetic acid is added 50 mg. of seleniumdioxide. The mixture is refluxed under nitrogen for 18 hours; then 50mg. of selenium dioxide is added and the mixture is rel uxed anadditional 24 hours. The product is filtered, and the filtrateevaporated to dryness. The residue is extracted with ethyl acetate andthe extract is washed successively with aqueous sodium bicarbonate,ammonium sulfide, dilute ammonia water, water, dilute hydrochloric acidand Water, and then dried over magnesium sulfate. The extract is treatedwith activated charcoal and then concentrated to dryness.Crystallization of the residue from a mixture of acetone and ether gives6a-methyl-17u-chloroethynyl-l ,4-androstadiene17fi-ol-3-one.

In accordance with the above procedures, but starting with the6a-methyl-17a-bromoethynyl-4-androstene-176- ol-3-one in place or" the6u-methyl-17a-cl1loroethynyl-4 androstene-17fi-ol-3-one, there isobtained the Get-methyl-17a-bromoethynyl-1,4-androstadiene-17B-ol-3-one.

Sepzomyxa afi'im's (ATCC 6,737) is inoculated from a slant into 250 ml.shake flasks containing 50 ml. of the following medium: 2% Edamin(Shefileld Farms), 5% glucose, and 0.5% corn steep liquor. After a 48hour incubation at 28 C., 10 mg. of6-methyl-17u-chloroethynyl-4-androstene-17,8-ol-3-one is added to eachshake flask as a dimethylformamide solution (100 mg./ml.). After aconversion period of 24 hours, the cells are removed by filtration,followed by three successive extractions with equal volume of ethylacetate. The extracts are combined and concentrated in vacuo.

The 6:: methyl 17a-chloroethynyl-1,4-androstadiene- 17fi-ol-3-one isreadily crystallized from the concentrate. Descending paperchromatography of the product in a system using formamide as thestationary phase and chloroform as the mobile phase, shows some of thesteroid substrate, but is largely the somewhat more polarA-dehydrogenation product.

Bacillus sphaericus (ATCC 12,488) is inoculated from a slant into 250ml. shake flasks containing 50 ml. of the following medium: 2% Edamin(Shefiield Farms), 5% glucose, and 0.5 corn steep liquor. After a 24hour incubation at 28 C., 10 mg. of6a-methyl-17a-chloroethynyl-4-androstene-17fi-ol-3-one is added to eachshake flask as a dimethylformamide solution (100 mg./ml.). After aconversion period of 24 hours, the cells are removed by filtration,followed by three successive extractions with equal volume of ethylacetate. The extracts are combined and concentrated in vacuo.

The 6st methyl 17a-chloroethynyl-1,4-androstadiene- 17B-ol-3-one, isreadilycrystallized from the concentrate. Descending paperchromatography of the product in a system using formamide as thestationary phase and chloro form as the mobile phase, shows some of thesteroid substrate, but is largely the somewhat more polar A'-dehydr0-genation product.

In accordance with the above procedures, but using theGa-methyl-17u-bromoethynyli-androstene-17,8-ol-3-0ne in place of the6u-methyl-17u-chloroethynyll-androstenel7B-ol-3-one, there is obtainedthe 6a-methyl-17a-bromoethynyl-1,4-androstadiene-17fl-ol-3-one.

Example 7 A suspension of 1 g. of Got-methyl-17-chloroethynyl-4-androstene-17fl-ol-3-one and 2.4 g. of chloranil in 30 ml. of ethylacetate and 6 ml. of acetic acid is stirred and refluxed for 16 hours.The reaction mixture is cooled and filtered. The product is washedsequentially with aqueous bisulfite, aqueous potassium hydroxide andWater. The organic layer is dried and concentrated in vacuo.Chromatography yields 6-methyl-17a-chloroethynyl-4,6-androstadiene-1713-01-3-one.

1 5 Example 8 To 100 mg. of6-methyl-17a-chloroethynyl-4,6-androstadiene-17/3-ol-3-one in 5 ml. oft-butanol and 0.1 ml. of acetic acid is added 50 mg. of seleniumdioxide. The mixture is refluxed under nitrogen for 18 hours; then 50mg. of selenium dioxide is added and the mixture is refluxed for anadditional 24 hours. The product is filtered and the filtrate evaporatedto dryness. The residue is extracted with ethyl acetate and the extractis washed successively with aqueous sodium bicarbonate, ammoniumsulfiide, dilute ammonia water, water, dilute hydrochloric acid andwater, and is then dried over magnesium sulfate. The extract is treatedwith activated charcoal and then concentrated to dryness.Crystallization of the residue from a mixture of acetone and ether gives6-methyl-17achloroethynyl-1,4,6-androstatriene-l7 8-ol-3-one.

In accordance with the above procedures, but starting with theG-methyl-17a-bromoethynyl-4,6-androstadiene- 17/3-ol-3-one in place ofthe 6-methyl-17a-chloroethynyl- 4,6-androstadiene-l7,8-01-3-one there isobtained the 6- methyl 17oz bromoethynyl 1,4,6-androstatriene-17,8-ol-3-one.

Sepzomyxa afim's (ATCC 6,737) is inoculated from a slant into 250 ml.shake flasks containing 50 ml. of the following medium: 2% Edamin(Sheflleld Farms), 5% glucose, and 0.5% corn steep liquor. After a 48hour incubation at 28 C., 10 mg. of6-met'hyl-l7a-chloroethynyl-4,6-androstadiene-l7,8-ol-3-one is added toeach shake flask as a dirnethylformamide solution (100 mg./ ml.). Aftera conversion period of 24 hours, the cells are removed by filtration,followed by three successive extractions with equal volume of ethylacetate. The extracts are combined and concentrated in vacuo.

The 6 methyl-17a-chloroethynyl-1,4,6-androstatriene- 17,8-ol-3-one, isreadily crystallized from the concentrate. Descending paperchromatography of the product in a system using formamide as thestationary phase and chloroform as the mobile phase, shows some of thesteroid substrate, but is largely the somewhat more polarA'-dehydrogenation product.

Bacillus sphaericus (ATCC 12,488) is inoculated from a slant into 250ml. shake flask containing 50 ml. of the following medium: 2% Edamin(Sheflield Farms), 5% glucose, and 0.5% corn steep liquor. After a24-hour incubation at 28 C., 10 mg. of6oc-methyl-l7a-chloroethynyl-4,6-androstadiene-17/3-ol-3-one is added toeach shake flask as a dimethylformamide solution (100 mg./m1.). After aconversion period of 24 hours, the cells are removed by filtration,followed by three successive extractions with equal volume of ethylacetate. The extracts are combined and concentrated in vacuo.

The Gar-methyl-17a-chloroethynyl-1,4,6 androstatriene- 17/3-ol-3-one, isreadily crystallized from the concentrate. Descending paperchromatography of the product in a system using formamide as thestationary phase, and chloroform as the mobile phase, shows some of thesteroid substrate, but is largely the somewhat more polarA'-dehydrogenation product.

In accordance with the above procedures, but starting with the6oc-methyl-17a-bromoethynyl-4,6-androstadiene- 17B-ol-3-one in place ofthe 16a-methyl-17a-ch1oroethynyl-4,6-androstadiene-17fi-ol-3-one, thereis obtained the 6a-methyl-l7a bromoethynyl 1,4,6 androstatriene-175-ol-3-one.

Example 9 p A mixture of 500 mg. of the6u-methyl-17a-chloroethynyl-4-androstene-17B-ol-3-one, 10 ml. ofdimethyl formamide, ml. of methyl iodide, and 1.5 gms. of silver oxideis stirred at room temperature for 4 days. An additional /z gm. ofsilver oxide is added at the end of each day. One hundred ml. ofchloroform is then added to the reaction mixture, then stirred for onehour, filtered, and the filtrate evaporated to dryness. The residual oilis chromatographed with acid-washed alumina and eluted Lil with mixturesof ether and petroleum ether to give 60:-methyl-17a-chloroethynyl-4-androstene 17,8 methoxy-3- one.

In accordance with the above procedures, but starting with the6a-methyl-17a-bromoethynyl-4-androstene-175- ol-3-one in place of the6a-methy1-17a-chloroethynyl-4- androstene-17fl-ol-3-one there isobtained the 6a-meth-yl-17a-bromoethynyl-4-androstene-17,8-methoxy-3-one.

mg. of Sa-methyl-l'7a-chloroethynyl-4-androstene- 17B-ol-3-one is heatedwith 1 cc. of acetic anhydride and 1.2 cc. of pyridine on the steam bathovernight. The reaction mixture is then poured onto ice and extractedwith chloroform. The extract is washed with water and concentrated. Theconcentrate is chromatognaphed over acid-Washed alumina and eluted withmixtures of ether and petroleum ether to give6cx-methyl-l7oc-chloroethynyl- 4-androstene-17.;3-01-3-one acetate.

In accordance with the above procedures, but starting with the6a-methyl-l7u-bromoethynyl-4-androstene-17,8- ol-3-one in place of the6oz-methyl-l7a-chloroethynyl-4- androstene-l7fi-ol-3-one there isobtained the 6a-methyl- 17a-brornoethynyl-4-androstene-17p-ol-3-oneacetate.

xample 10 100 mg. of i7a-chloroethynyl-4-androstene-176-ol-3-one(Example 1) is heated with 1 cc. of acetic anhydride and 1.2 cc. ofpyridine on the steam bath overnight. The reaction mixture is thenpoured onto ice and extracted with chloroform. The extract is washedwith water and concentrated. The concentrate is chromatographed overacid-washed alumina and eluted with mixtures of etherpetroleum and etherto give 17a'chloroethynyl-4-androstene-17fi-ol-3-one acetate.

In accordance with the above procedure, but starting with thel7a-bromoethynyl-4-androstene-17 3-ol-3-one in place of the17a-chloroethynyl-4-androstene-17flol-3-one there is obtained thel7a-bromoethynyl-4-androstene-175- ol-3-one acetate.

Example 11 To 100 mg. of 17or-chloroethynyl-4-androstene-175-01- 3-oneacetate in 5 ml. of t-butanol and 0.1 ml. of acetic acid is added 50 mg.of selenium dioxide. The mixture is refluxed under nitrogen for 18hours; then 50 mg. of selenium dioxide is added and the mixture isrefluxed an additional 24 hours. The product is filtered, and thefiltrate evaporated to dryness. The residue is extracted with ethylacetate and the extract is washed successively with aqueous sodiumbicarbonate, water, dilute hydrochloric acid and water, and then driedover magnesium sulfate. The extract is treated with activated charcoaland then concentrated to dryness. Crystallization of the residue gives17a-chloroethynyl-1,4-androstadiene-l713-ol- 3-one acetate.

In accordance with the above procedures, but starting with the17a-bromoethynyl-4-androstene-17,5 ol 3 one acetate in place of the17a-chloroethynyl-4-androstene- 17B-ol-3-one acetate, there is obtainedthe 17a-bromoethynyl-1,4-androstadiene-17fi-ol-3-one acetate.

Corynebacterium simplex (ATCC 6,946) is inoculated from a slant to 250ml. of shake flasks containing a medium having the composition: 1g./liter yeast extract (Difco). After an 18 hour growth phase at 28 C.,10 mg. of 17a-chloroethynyl-4-androste-ne-17fi-ol-3-one acetate is addedto each flask as a dimethylformamide solution (100 mg./ml.). After a24-hour transformation period at 28 C., the cells are centrifuged,followed by three ethyl acetate extracts of the cell-free broth. Theextracts are combined and concentrated in vacuo. The17a-chloroethynyl-L4- androstadiene-l7,8-01-3-one acetate iscrystallized directly from the concentrate. Paper chromatography of theproduct in a system utilizing formamide as the stationary phase andchloroform-benzene (1:1) as .the mobile phase indicates that the productpossesses a polarity slightly greater than the substrate.

.Scptomyxa afiinls (ATCC 6,737) is inoculated from a slant into 250 ml.shake flasks containing 50 ml. of the following medium: 2% Edamin(Sheiiield Farms), 5% glucose, 0.5% corn steep liquor. After a 48-hourincubation at 28 C., mg. of 17a-chloroethynyl-4-androstene- 17B-ol-3-oneacetate is added to each shake flask as a dimethylfornramide solution(100 mg./ml.). After a conversion period of 24 hours, the cells areremoved by filtration, followed by three successive extractions withequal volume of ethyl acetate. The extracts are combined andconcentrated in vacuo.

The product is re-acetylated by treatment with acetic anhydride andpyridine, and t en purified by recrystallization to give the17a-chloroetnynyl-1,44androstadiene-17fiol-3-one acetate. Descendingpaper chromatogaphy of the product in a system using formamide as thestationary phase, and chloroform as the mobile phase, shows some of thesteroid substrate, but is largely the somewhat more polarA-dehydrogenation product.

In accordance with the above procedures, but using the17oz-bromocthynyll-androstene-17B-ol-3-one acetate in place of the17u-chloroethynyl-4-androstene-l7fl-ol-3 one acetate, there is obtainedthe l7a-br0moethynyl-L4- androstadiene-l7fi-ol-3-one acetate.

Example 12 A mixture of 500 mg. of the 17a-chlor0ethynyl-4-androstene-17 8-ol-3-one, 10 ml. of dimethylformamide, ml. of methyliodide, and 1.5 gms. of silver oxide is stirred at room temperature for4 days. An additional /2 gm. of silver oxide is added at the end of eachday. Gne hundred of chloroform is then added to the reaction mixture,which is then stirred for one hour, filtered, and the filtrate isevaporated to dryness. The residual oil is chromatographed withacid-washed alumina and eluted with mixtures of ether and petroleumether to give l7a-chloroethynyl-4-androstene-17B-methoxy-3- one.

In accordance with the above procedures, but starting with the17m-bromoethynyl-4-and-rostene-17/3-ol-3-one in place of thel7a-chloroethynyl-4-androstenc-1719-01-3-one, there is obtained the17a-bromoethynyl-4-androstenc-l7B- methoxy-3-one.

Example 13 To 100 mg. of 17u-chloroethynyl-4-androstene-17,8-methoxy-3-one in 5 ml. of t-butanol and 0.1 ml. of acetic acid is addedmg. of selenium dioxide. The mixture is refluxed under nitrogen for 18hours; then 50 mg. of selenium dioxide is added and the mixture isrefluxed an additional 24 hours. The product is filtered, and thefiltrate evaporated to dryness. The residue is extracted with ethylacetate and the extract is washed successively with aqueous sodiumbicarbonate, water, dilute hydrochloric acid and water, and then driedover magnesium sulfate. The extract is treated With activated charcoaland then concentrated to dryness. Crystallization of the residue from amixture of acetone and ether gives 17a chloroethynyl -1,4androstadiene-UB-methox 3-one.

Sepzomyxa afiinis (ATCC 6,737) is inoculated from a slant into 250 m1.shake flasks containing 50 ml. of the following medium: 2% Edamin(Shefiield Farms), 5% glucose, and 0.5% corn steep liquor. After a 48hour incubation at 28 C., 10 mg. of l7ct-chloroethynyl-4-androstene-17,8-methoxy-3-one is added to each shake flask as adimethylformamide solution (100 mg./rr1l.). After a conversion period of24 hours, the cells are removed by filtration, followed by threesuccessive extractions with equal Volume of ethyl acetate. The extractsare combined and concentrated in vacuo.

The 17u-chloroethynyl-1,4-androstadiene-17,8-methoxy- 3-one is readilycrystallized from the concentrate. Descending paper chromatography ofthe product in a system using formamide as the stationary phase andchloroform as the mobile phase, shows some of the steroid 118 substrate,but is largely the somewhat more polar A-dehydrogenation product.

Bacillus spl'laericus (ATCC 12,488) is inoculated from a slant into 250ml. shake flasks containing 50 ml. of the following medium: 2% Edamin(Sheffield Farms), 5% glucose, and 0.5% corn steep liquor. After a 24hour incubation at 28 C., 10 mg. of 17 a-chloroethynyl-4-androstene-l7fi-methoxy-3-one is added to each shake flask as adimethylformamide solution mg./ml.). After a conversion period of 24hours, the cells are removed by filtration, followed by three successiveextractions with equal volume of ethyl acetate. The extracts arecombined and concentrated in vacuo.

The 17u-chloroeth-yny1-1,4-androstadiene-l7fi-methoxy- 3-one, is readilycrystallized from the concentrate. Descending paper chromatography ofthe product in a system using formamide as the stationary phase andchloroform as the mobile phase, shows some of the steroid substrate, butis largely the somewhat more polar A-dehydrogenation product.

In accordance with the above procedures, but using the 170: bromoethynyl4-androstene-17B-methoxy-3-one in place of the17a-chloroethynyl-4-androstene-17,8- methoxy-3-one, there is obtainedthe 17a-br0moethynyl 1,4-androstadieue-l7B-methoxy-3-one.

Example 14 A mixture of 1 g. of l7a-chloroethynyl-4-androstene-17B-ol-3-one, 10 ml. of acetic anhydride and 100 mg. ofp-toluenesulfonic acid is heated on the steam bath for one hour andallowed to stand overnight at room temperature. It is then poured intoice water and left to stand at room temperature for one half hour. Thereaction mixture is then extracted with ether. The ether extract iswashed with water and aqueous sodium bicarbonate, dried, andconcentrated to yield the crude 17t1-Ch101'0-cthy'nyl-Ei,5-androstadiene-3,175-dio l diacetate. The prodduct,crystallized from petroleum ether, has a melting point of l3l C. Theanalysis of a sample which has a melting point of 136 C. is: C, 69.75;7.23. Calculated C, 69.67; H, 7.25.

To a solution of 200 mg. of of the 17a-chloroethynyl-3,5-androstadiene-3,17,8-diol-diacetate dissolved in 2 ml. of aceticacid is added with stirring, 56 ml. of N-chlorosuccinirnide and 2 ml. oftetrahydrofuran containing 5% of dry HCl. The reaction is stirred atroom temperature for 2 /2 hours. Sodium bicarbonate is added and thereaction mixture is extracted with ether. The ether extracts are driedand concentrated. The material is chromatographed over acid-washedalumina, and eluted with ether-petroleum ether mixtures, to give the60:- chloro- 17 a-chloroethynyl-4-androstenel7fi-ol-3-one acetate. Theanalytical sample is crystallized from a mixture of methylenechloride-ether and has the following properties: Ml. 220-230 C.

M3181? 235 In e 13,600

Arzalysis.-Calculated: C, 65.26; H, 6.67; CI, 16.75. Found: C, 65.80; H,6.74; Cl, 17.86.

in accordance with the above procedures, but starting with the17a-bromoethynyl-4-androstene-l7B-ol-3-one in place of the17wchloroethynyl-4-androstene-175-01-3- one, there is obtained asintermediate, thel7u-b-romoethynyl-3,5-androstadiene-3,l7,8-diol-diacetate, and asproduct, the 6a-chloro-17a bromoethynyl-4-androstene- 17,8-ol-3-oneacetate.

Example 15 To 100 mg. of6a-chloro-17a-chloroethynyl-4-androstene-17fi-ol-3-one acetate in 5 ml.of t-butanol and 0.1 m1. of acetic acid is added 50 mg. of seleniumdioxide. The mixture is refluxed under nitrogen for 18 hours; then 50mg. of selenium dioxide is added and the mixture is refluxed anadditional 24 hours. The product is filtered, and the filtrateevaporated to dryness. The residue is extracted with ethyl acetate andthe extract is Washed successively lid with aqueous sodium bicarbonate,ammonium sulfide, dilute ammonia water, water, dilute hydrochloric acidand water, and then dried over magnesium su fate. The extract is treatedwith activated charcoal nd then concentrated to dryness. Crystallizationof the residue from a mixture of acetone and ether gr es6o:-Clll6lO-17oc-ClllGTO ethynyl-l,4-androstadiene-l7l3-ol-3-oneacetate.

In accordance with the above procedures, but starting with theoa-chloro-l7a-bromoethynyl-4-androstene-17,8- ol-3-one acetate in placeof the om-chloro-i'ia-chloroethynyl-4-androstene-l7fi-ol-3-one acetatethere is obtained the 6 Cl1l0f0"l7OL bZ'QHlGBthEIIE/l-1,4-afidr0stadlene-l7fl-ol-3-one acetate.

Corynebacterimn simplex (ATCC 6,946) is inoculated from a slant to 250ml. shake flasks contai a medium having the composition: 1 g./llteryeast extract (Difco). After an 18 hour growth phase at 28 C., 10 mg. of60acnloro-l7a-ehloroethynyl-4-androstene-l7,6-ol-3-one acetate is addedto each flask as a dim-ethylformamide solution (100 rug/ml). After a 24hour transformation period at 28 C., the cells are centrifuged, followedby three ethyl acetate extracts of the cell-free broth. The extracts arecombined and concentrated in vacuo. T he 6e-chlorol7a-chloroethynyl-1,4-androstadiene-17,8-ol-3-one acetate is crystallizeddirectly from the concentrate. Paper chromatography of the product is asystem utilizing formamide as the stationary phase andchloroform-benzene (1: l) as the mobile phase indicates that the productposse ses a polarity slightly greater than the substrate.

Septomyxa aflinis (ATCC 6,737) is inoculated from a slant into 250 shakeflasks containing 50 m1. of the following medium: 2% Edarnin (SheffieldFarms), 5% glucose, and 0.5% corn steep liquor. After a 48 hourincubation at 28 C., mg. of 6oL-Cl'1lOI'O-l7oc-Cl1l0l0-ethynyll-androstene-17fl-ol-3-one acetate is added to each shake flaskas a dimethylformamide solution (100 mg./ ml.). fter a conversion periodof 24 hours, the cells are removed by filtration, followed by threesuccessive extractions with equal volume of ethyl acetate. The extractsare combined and concentrated in vacuo.

The product is re-acetylai'ed by treatment with acetic anhydride andpyridine, and then purified by recr staliiza- (ion to give the6d-chloro-l7a-chloroethynyl-1,4-androstadlene-l7fi-ol-3-one acetate, isreadily crystallized from the concentrate. Descending paperchromatography of the product in a system using amide as the stationaryphase and chloroform as the mobile phase, shows some of the steroidsubstrate, but is largely the somewhat more polar A'-dehydrogenationproduct.

In accordance with the above procedures, but using the 60: chloro17a-brornoethynyl-4-androstene-l7fi ol-3-one acetate in place of th6a-chloro-17a-chloroethynyl-4-androstene-17fl-ol-3-one acetate, there isobtained the 6dchloro 17a-bromoethynyl-l,4-androstadiene-l75-ol-3-oneacetate.

Example 16 A mixture of 1 g. of 17a-chloroethynyl-4-androstene-17,5-methoxy-3-one (Example 10), 10 ml. of acetic anhydride and 190 mg.of p-toluenesulfonic acid is heated on the steam bath for one hour andallowed to stand overnight at room temperature. It is then poured intoice Water and left to stand at room temperature for one half hour. Thereaction mixture is en extracted with ether. The ether extract is washedWlLh water and aqueous sodium bicarbonate, dried, and concentrated toyield the 17cc chloroethynyl 3,5-androstadiene-l7/3-methoxy-3-olacetate.

To a solution of 260 mg. of the17u-chloroethynyl-3jandrostadiene-175-methoxy-3-ol-acetate dissolved in2 ml. of acetic acid is added, with stirring, 56 ml. of N-chloro-'succinimide and 2 ml. of tetrahydrofuran containing 5% of dry HCl. TLereaction is stirred at room temperature for 2 /2 hours. Sodiumbicarbonate is added. The reaction mixture is then extracted with ether.other extracts are dried and concentrated. The material is chro- Example17 To mg. of 17o:-chloroethynyl-4-androstene-175- methoxy-3-one in 5 ml.of t-butanol and 0.1 ml. of acetic acid is added 50 mg. of seleniumdioxide. The mixture is under nitrogen for 18 hours; then 50 mg. ofselenium dioxide is added and the mixture is refluxed an additional 24hours. The product is filtered, and the liltrate evaporated to dryness.The residue is extracted with ethyl acetate and the extract is washedsuccessively with aqueous sodium bicarbonate, ammonium sulfide, diluteammonia Water, water, dilute hydrochloric acid and water,

and then dried over magnesium sulfate. the extract is treated withactivated charcoal and then concentrated to dryness. Crystallization ofthe residue from a mixture of acetone and ether givesl7a-chloroethynyl-l,4-androstadiene-l7fi-methoxy-3-one.

Septomyxa affim's (ATCC 6,737) is inoculated from a slant into 25-0 ml.shake flasks containing 50 ml. of the following medium: 2% Edamin(Sheffield Farms), 5% glucose, and 0.5% corn steep liquor. After a 48hour incubation at 28 C., 10 mg. of l7cx-cl'iloroethynyl4-androstene-l'ifi-methoxyJ-one is added to each hake flask as adimethylformamide solution (100 rug/ml). After a conversion period of 24hours, the cells are removed by filtration, followed by three successiveextractions with equal volume of ethyl acetate. The extracts arecombined and concentrated in vacuo.

The 17u-chloroethynyl-1,4-androstadiene-l75unethoxy- 3-one is readilycrystallized from the concentrate. Descending paper chromatography ofthe product in a system using formamide as the stationary phase andchloroform as the mobile phase, shows some of the steroid substrate, butis largely the somewhat more polar A-dehydrogenation product. a

Bacillus splmericus (ATCC 12,488) is inoculated from a slant into 250ml. shake flasks containing 50 ml. of the following medium: 2% Edamin(Shelficld Farms), 5% glucose, and 0.5% corn steep liquor. After a 24hour incubation at 28 C., 10 mg. of17a-chloroethynyl-4-androstene-l7,8-methoxy-3-one is added to each shakeflask as a dimethylronnamide solution mg./ml.). After a conversionperiod of 24 hours, the cells are removed by' as the mobile phase, showssome of the steroid substrate, i but is largely the somewhat more polard dehydrogena tion product.

In accordance with the above procedures, but using the bromoethynyl 4androstene-l7,B-methoxy-3-one in a I place or" the 17a-chloroethynyl--androstene-l7fi-methox 3-one, there is obtained the17a-b'romoethynyl-1,4-androstadiene-17,3-methoxy-3-one.

Example 18 A suspension or" 1 g.

lluxed for 16 hours. 'lhe reaction mi. are is cooled and filtered. prodct is Washed sequentially with aqueous of 17echloroethynyl lendro V:stene-l7,8ol-3-one and 2.4 g. of chloranilin 36 m1. of ethyl acetate and6 ml. of acetic acid is stirred and re-' bisulfite, aqueous potassiumhydroxide and Water. Chromatography yields17a-chloroethynyl-4,G-androstadiene- 17fi-ol-3-one which oncrystallization from ethyl acetate has the following properties: M.P.209-21l C:

Found:

U.V. R252 240 In;;, E% 377 A solution of 140 mg. of the oxide in m1. of0.4 N hydrogen chloride in chloroform is allowed to stand at roomtemperature for 5.5 hours. The solution is poured onto ice. Aqueoussodium bicarbonate solution is added and the product is extracted withchloroform. The organic layer is washed with Water, dried andconcentrated. Crystallization from ether affords 84 mg. of 6-chloro-l7-chloroethynyl-4,6-androstadiene-l7/3-ol-3-one, MP. 195 205 C. The samplefor analysis is crystallized from methanol and has the followingproperties: MP. 203- 208 C; -96 (C 1.0 CHCl U.V. its? 285 m 6 21,400

Calculated for C H O Cl C, 66.49; H, 6.38. C, 66.28; H, 6.62.

In accordance with the above procedures, but starting with the17ct-bronioethynyli-androstene-17(3-ol-3-one in place of the 17u-chloroethynyl-4-androstene-17,8-ol-3-one, there is obtained asintermediate, the Nix-bromoethynyl-4-androstene-l7fi-ol-3-one-6,7-oxide, and as product, the6-chloro17-bromoethynyl-4,6androstadiene-17/3-ol-3-one.

A solution of 125 mg. of 6-chloro-l7a-chloroethynyl-4,6-androstadiene-175-ol-3-one in 1.8 ml. of pyridine and 1.5 ml. ofacetic anhydride is heated on the steam bath overnight. The solution ispoured into ice water and extracted with ether. The ether solution iswashed sequentially with dilute hydrochloric acid, water and sodiumbicarbonate solution. Chromatography on 7.0 g. of acid- Found:

washed alumina, and elution with ether; petroleum ether mixtures affords56 mg. of 6-chloro-l7a-chioroethynyl- 4,6-androstadiene-l7fi-ol-3-oneacetate. The sample for analysis is crystallized from ether and has thefollowing properties :M.P. 203-208 C., a 98 (C, 0.9 CHCig);

U.V. H552 284 my, e 21,300

Calculated for C H O Cl C, 65.57; H, 6.21. C, 66.06; H, 6.16.

In accordance with the above procedures, but starting with the 6chloro-17a-bromoethynyll,6-androstadienel7 8-ol-3-one in place of the6-chloro-l7u-chloroethynyl- 4,6-".ndrostadiene17fi'ol-3-one, there isobtained the 6- chloro 171x bromoethynyl-4,6-androstadiene-l7fl-ol-3-one acetate.

Found Example 19 283 Inn e 26900 5 22 nesium sulfate. The extract istreated with activated charcoal and then concentrated to dryness.Crystallization of the residue gives 6-chloro-17x-chloroethynyl-1,4,6-androstatriene-17B-ol-3-one acetate.

Corynebacterium simplex (ATCC 6,946) is inoculated from a slant to 250ml. shake flasks containing a medium having the composition: 1 g./literyeast extract (Difco). After an 18 hour growth phase at 28 C., 10 mg. of6-chloro-l7a-ch10roethynyl-4,6-androstadienel7fi-ol-3-one acetate isadded to each flask as a dimethylformamide solution rug/ml). After a 24hour transformation period at 28 C., the cells are centrifuged, followedby three ethyl acetate extracts of the cell-free broth. The extracts arecombined and concentrated in vacuo. The6-chloro-l7a-chloroethynyl-1,4,6-androstatriene-17B-ol-3-one acetate iscrystallized directly from the concentrate. Paper chromatography of theproduct is a system utilizing formamide as the stationary phase andchloroform-benzene (1:1) as the mobile phase indicates that the productpossesses a polarity slightly greater than the substrate.

Septomyxa afitnis (ATCC 6,737) is inoculated from a slant into 250 ml.shake flasks containing 50 m1. of the following medium: 2% Edamin(Shefield Farms), 5% glucose, and 0.5% corn steep liquor. After a 48hour incubation at 28 C., 10 mg. of 6-Cl11OI0-17oc-Ch101'0-ethynyl-4,6-androstadiene-17B-ol-3-one acetate is added to each shakeflask as a dimethylformamide solution (100 mg./ml.). After a conversionperiod of 24 hours, the cells are removed by filtration, followed bythree successive extractions with equal volume of ethyl acetate. Theextracts are combined and concentrated in vacuo.

The product is re-acetylated by treatment with acetic anhydride andpyridine, and then purified by recrystallization to give the6-chloro-l7ot-chloroethynyl-1,4,6-androstatriene-17,8-ol-3-one acetate.Descending paper chromatography of the product in a system usingformamide as the stationary phase and chloroform as the mobile phase,shows some of the steroid substrate, but is largely the somewhat morepolar A'-dehydrogenation product.

In accordance with the above procedures, but using the 6 chloro 17o.bromoethynyl 4,6 androstadiene- 17,8-ol-3-one in place of the6-chloro-l7a-chloroethynyl- 4,6-androstadiene-l7B-ol-3-one, there isobtained the 6- chloro 171x bromoethynyl 1,4,6 androstatriene 1713-ol-3-one acetate.

Example 20 A mixture of 500 mg. of 6-chloro-17a-chloroethynyl-4,6-androstadiene-17,8-01-3-one, 10 ml. of dimethylformamide, 20 ml. ofmethyl iodide, and 1.5 gms. of silver oxide is stirred at roomtemperature for 4 days. An additional /2 gm. of silver oxide is added atthe end of each day. 100 ml. of chloroform is then added to the reactionmixture which is then stirred for one hour, filtered. The filtrate isthen taken to dryness. The oil is chromatographed with acid-Washedalumina and eluted with mixtures of ether and petroleum ether to givethe 6 chloro 17a chloroethynyl 17B methoxy 4,6 androstadiene-3-one.

In accordance with the above procedures, but starting with the6-chloro-17a-bromoethynyl-4,6-androstadiene- 17,8-ol-3-one in place ofthe 6-chloro-17a-chloroethynyl- 4,6-androstadiene-17fi-ol-3-one there isobtained the 6 chloro 17oz bromoethynyl 17,8 methoxy 4,6androstadiene-3-one.

Example 21 To 100 mg. of 6 .chloro 17cc chloroethynyl 17/8-rnethoxy-4,6-androstadiene-3-one in 5 ml. of t-butanol and 0.1 ml. ofacetic acid is added 50 mg. of selenium dioxide. The mixture is refluxedunder nitrogen for 18 hours; then 50 mg. of selenium dioxide is addedand the mixture is refluxed an additional 24 hours. The product isfiltered, and the filtrate evaporated to dryness. The residue isextracted with ethyl acetate and the extract is washed successively withaqueous sodium bicarbonate, ammonium sulfide, dilute ammonia water,water, dilute hydrochloric acid and Water, and then dried over magnesiumsulfate. The extract is treated with activated charcoal and thenconcentrated to dryness. Crystallization of the residue from a mixtureof acetone and ether gives the6-chloro-17a-chloroethynyl-175-methoxy-1,4,6- androstatriene-3-one.

Septomyxa afiinis (ATCC 6,737) is inoculated from a slant into 256 ml.shake flasks containing 50 ml. of the following medium: 2% Edarnin(Sheflield Farms), glucose, and 0.5% corn steep liquor. After a 48 hourincubation at 28 C., mg. of6-chloro-l7a-chloroethynyl-17B-methoxy-4,6-androstadiene-3-one is addedto each shake flask as a dimethylformamide solution (100 mg./ml.). Aftera conversion period of 24 hours, the cells are removed by filtration,followed by three successive extractions with equal volume of ethylacetate. The extracts are combined and concentrated in vacuo.

The 6 chloro 17a chloroethynyl 17,8 methoxyl,4,6-androstatriene-3-one isreadily crystallized from the concentrate. Descending paperchromatography of the product in a system using formamide as thestationary phase and chloroform as the mobile phase, shows some of thesteroid substrate, but is largely the somewhat more polarA'-dehydrogenation product.

Bacillus sphaericus (ATCC 12,488) is inoculated from a slant into 250ml. shake flasks containing 50 m1. of the following medium: 2% Edamin(Sheffield Farms), 5% glucose, and 0.5% corn steep liquor. After a 24hour incubation at 28 C., 10 mg. of6-chloro-l7a-ch1oroethynyl-4,6-androstadiene-17,6-rnethoxy-3-one isadded to each shake flask as a dimethylformamide solution (100 mg./ml.).After a conversion period of 24 hours, the cells are removed byfiltration, followed by three successive extractions with equal volumeof ethyl acetate. The extracts are combined and concentrated in vacuo.

The 6 chloro 17a chloroethynyl 17,8 rnethoxy- 1,4,6-androstatriene-3-oneis readily crystallized from the concentrate. Descending paperchromatography of the product in a system using formamide as thestationary phase and chloroform as the mobile phase, shows some of thesteroid substrate, but is largely the somewhat more polarA'-dehydrogenation product.

In accordance with the above procedures, but using the 6 chloro 17abromoethynyl 4,6 androstadienel7 3-methoxy-3-one in place of the6-chloro-17a-chloroethynyl-4,6-androstadiene-17fi-methoxy-3-one, thereis obtained the 6 chloro 17c: bromoethynyl 1,4,6 androstatriene- 17,S-methoxy-S-one.

Example 22 A solution of the 17u.-ch:ioroethynyl-3,S-androstadiene-3,8,17,8-diol diacetate (Example 1%) in aqueous tetrahydrofuran istreated with a slow stream of perchloryl fi ide for one hour and thenallowed to stand at room temperature for an additional 2 hours. Thereaction mixture is poured on ice and extracted with chloroform. Thechloroform layer is washed with aqueous bicarbonate solution, dried andconcentrated vacuo. The crude material is dissolved in acetic acidsaturated with gaseous hydrogen chloride and allowed to stand for onehour at room temperature.

F ad fluoro 17a bromoethynyl 4 androsten 173 ol- 3-one acetate.

Example 23 To mg. of a-fiuoro-l7e-chloroethynyl-4-androstene-l7fi-ol-3-one acetate in 5 ml. of t-butanol and 0.1 ml. of acetic acid isadded 50 mg. of selenium dioxide. The mixture i refluxed under nitrogenfor 18 hours; then 50 mg. of selenium dioxide is added and the mixtureis refluxed an additional 24 hours. The product is filtered, and the filrate evaporated to dryness. The residue is extracted with ethyl acetateand the extract is washed successively with aqueous sodium bicarbonate,water, dilute hydrochloric acid and water, and then dried over magnesiumsulfate. The extract is treated with activatcd charcoal and thenconcentrated to dryness. Crysallization of t..e residue from a mixtureof acetone and ether gives6ot-fluoro-l7ct-ohloroethynyl-1,4-androstadiene- 17,3-oi-3-one acetate.

Coryizebacterium simplex (ATCC 6,946) is inoculated from a slant to 250ml. shake flasks containing a medium having the composition: 1 g./literyeast extract (Difco). After an 18 hour growth phase at 28 C., 10 mg. of6afluoro 17cc chloroethynyl 4 androstene 173 o"- 3-one acetate is addedto each flask as a diniethylformamide solution (100 gm./ml.). After a 24hour transformation period at 28 (3., the cells are centrifuged,followed by three ethyl acetate extracts of the cell-free broth. Theextracts are combined and concentrated in vacuo. The. 6ufluoro-17a-chloroethynyl-1,4-androstadiene-l7fl-ol-3-oue acetate iscrystallized directly from the concentrate. Paper chromatography of theproduct is a system utilizing formamide as the stationary andchloroform-benzene (1:1) as the mobile phase indicates that the productpossesses a polarity slightly greater than the substrate.

Seplomyxa affim's (ATCC 6,737) is inoculated froma slant into 250 ml.shake flasks containing 56 ml. of the following medium: 2% Edarnin(Sheffield Farms), 5 glucose, and 0.5% corn steep liquor. .fter a 48hour incubation at 28 C., 10 mg. of6.x-fiuoro-17e-chloroethynyl-4-androstene-17,8-ol-3-one acetate is addedto eac.

shake flask as a dimetlhylform'amide solution (100 mg].

ml). After a conversion period of 24 hours, the cells are removed byfiltration, followed by three successive extractions with equal volumeof ethyl acetate. The extracts are combined and concentrated in vacuo.

The product is re-acetylated by treatment with acetic; anhydride andpyridine, and then purified by recrystallination to give the6a-fiuoro-l7c-chloroeth3myl-1,4-androstadiene-17fi-ol-3-one acetate.Descending paper chromatography of the product in a system usingformarnide as the stationary phase and chloroform as the mobile phase,shows some of the steroid substrate, but is largely the somewhat morepolar N-dehydrogenation product.

in accordance with the above procedures, but using the 6d fiuoro 17:1bromoethynyl 4 androstene 17f3- ol-3-one acetate in place of the6a-fiuoro-l7a-chloro:

ethynyl-4-androstere-17p3-ol-3-one acetate there is obtained theGet-fluoro-l7c-bromoethynyl-1,4-androstadiene l7,8-ol-3-one acetate.

Example 24 A suspension of 1 g. of a-fluoro-17a-chloroetbynyl 4-androstene-l7/3-ol-3-one acetate 2.4 g. chlorani-l, '30 ml;

in accordance with the above procedures, but starting with the6a-fluoro-17a bromoethynyl-4-androstene-176-ol- 3 one acetate in placeof the Get-fluoro-l7ot-chloroethynyl- 4-androstene-l7 S-ol-one acetatethere is obtained'the 6:-

lue product is washed sequentially with aque- 25 fiuoro 17a bromoethynyl4,6 androstadiene 17B- ol-3-one acetate.

Example 25 To 100 mg. of6-fluoro-17a-chloroethynyl-4,6-androstadiene-17fi-ol-3-one acetate in 5ml. of t-butanol and 0.1 ml. of acetic acid is added 50 mg. of seleniumdioxide. The mixture is refluxed under nitrogen for 18 hours; then 50mg. of selenium dioxide is added and the mixture is refluxed anadditional 24 hours. The product is filtered, and the filtrateevaporated to dryness. The residue is extracted with ethyl acetate andthe extract is washed successively with aqueous sodium bicarbonate,water, dilute hydrochloric acid and water, and then dried over magnesiumsulfate. The extract is treated with activated charcoal and thenconcentrated to dryness. Crystallization of the residue givesG-fluoro-l7a-chloroethynyl-1,4,6- androstatriene-17fi-0l-3-one acetate.

in accordance with the above procedures, but starting with the6-fluoro-l7a4bromoethynyl-4,6-androstadiene- 17B-ol-3-one acetate inplace of the 6-fluoro-17x-chloroet-hynyl-4,6-androstadiene-l7fl-ol-3-one acetate, there isobtained theo-fluoro-17a-bromoethynyl-1,4,6-androstatriene-l7fi-ol-3-one acetate.

Coryizebacterizlm simplex (ATCC 6,946) is inoculated from a slant to 250ml. shake flasks containing a medium having the composition: 1 g./literyeast extract (Difco). After an 18 hour growth phase at 28 C, mg. of 6-fluoro-17u-chloroethynyl-4,6-androstadiene 175 ol 3- one acetate isadded to each flask as a dimethylformamide solution (100 mg./ml.). Aftera 24 hour transformation period at 28 C., the cells are centrifuged,followed by three ethyl acetate extracts of the cell-free broth. Theextracts a e combined and concentrated in vacuo. The6-fluoro-17a-chloroethynyl-1,4,6-androstatriene 175 ol- 3-one acetate iscrystallized directly from the concentrate. Paper chromatography of theproduct in a system utilizing formamide as the stationary phase andchloroform-benzene (1:1) as the mobile phase indicates that the productpossesses a polarity slightly greater than the substrate.

Septomyxa aflfnis (ATCC 6,737) is inoculated from a slant into 250 ml.shake flasks containing 50 ml. of the following medium: 2% Edamin(Sheffield Farms) 5% glucose, and 0.5% corn steep liquor. After a 48hour incubation at 28 C., 10 mg. of6-fluoro-17a-chloroethynyl-4,6-androsiadiene-17-ol-3-one acetate isadded to each shake flask as a dimethylformamide solution (100 mg./ml.). After a conversion period of 24 hours, the cells are removed byfiltration, followed by three successive extractions with equal volumeof ethyl acetate. The extracts are combined and concentrated in vacuo.

The product is re-acetylated by treatment with acetic anhydride andpyridine, and then purified by recrystallization to give the6a-fluoro-17a-chloroethynyl-1,4,6-androstatriene-UB-ol-S-one acetate.Descending paper chromatography of the product in a system usingformamide as the stationary phase and chloroform as the mobile phase,shows some of the steroid substrate, but is largely the somewhat morepolar A'-del1ydrogenation product.

In accordance With the above procedures, but using theG-fluoro-17:z-bromoethynyl-4,6-androstadiene 17,8 01-3- one acetate inplace of the 6-fluoro-17a-chloroethynyl 4-,6-androstadiene-17B-ol-3-oneacetate, there is obtained the6-fluoro-17a-bromoethynyl-1,4,6-androstatriene 1713- ol-3-one acetate.

Example 26 A solution of thel7a-chloroethynyl-3,5-androstadienel7fl-methoxy-3fi-ol in aqueoustetrahydrofuran is treated with a slow stream of perchloryl fluoride forone hour and then allowed to stand at room temperature for an additional2 hours. The reaction mixture is poured on ice and extracted withchloroform. The chloroform layer is Washed with aqueous bicarbonatesolution, dried and concentrated in vacuo. The crude material isdissolved in acetic acid saturated with gaseous hydrogen chloride andallowed to stand for one hour at room temperature. The reaction mixtureis poured on ice and extracted with chloroform. The chloroform layer iswashed with aqueous bicarbonate solution, dried and concentrated invacuo. The concentrate is chromatographed on acid-washed alumina toyield the 6a-fluor0-17a-chl0r0- ethynyl-4-androstene-l7B-methoxy-3-one.

In accordance with the above procedures, but starting with the17a-bromoethynyl-3,S-androstadiene-l7fi-methoxy-Iifi-ol in place of the17a-chloroethynyl-3,S-androstadiene-17(3-methoxy-3B-ol, there isobtained the Get-fillorol7u-bromoethynyl-4-androstene-17fi-methoxy-3-one.

Example 27 To mg. of6u-fluoro-17a-chloroethyny1-4-androstene-l7fi-methoxy-3-one in 5 ml. oft-butanol and 0.1 ml. of acetic acid is added 50 mg. of seleniumdioxide. The mixture is refluxed under nitrogen for 18 hours; then 50mg. of selenium dioxide is added and the mixture is refluxed anadditional 24 hours. The product is filtered, and the filtrateevaporated to dryness. The residue is extracted with ethyl acetate andthe extract is Washed successviely with aqueous sodium bicarbonate,water, dilute hydrochloric acid and water, and then dried over magnesiumsulfate. The extract is treated with activated charcoal and thenconcentrated to dryness. Crystallization of the residue gives6ot-fluoro-17a-chloroethynyl-1,4- androstadiene-17fi-n1ethoxy-3-one.

Septomyxa ayjfinis (ATCC 6,737) is inoculated from a slant into 250 ml.shake flasks containing 50 ml. of the following medium: 2% Edamin(Sheffield Farms), 5% glucose, and 0.5% corn steep liquor. After a 48hour incubation at 28 0., 10 mg. of 6st-fillOI'O-17a-Cl2l0l'0-ethynyl-4-androstene-17B-methox -3-one is added to each shake flask as adimethylformamide solution (100 mg./ ml). After a conversion period of24 hours, the cells are removed by filtration, followed by threesuccessive extractions with equal volume of ethyl acetate. The extractsare combined and concentrated in vacuo.

The 6a-flu0ro-17a-chloroethynyl-1,4-androstadiene-17B- methoxy-S-one isreadily crystallized from the concentrate. Descending paperchromatography of the product in a system using formamide as thestationary phase and chloroform as the mobile phase, shows some of thesteroid substrate, but is largely the somewhat more polar A'-dehydrogenation product.

Bacillus sphaericzls (ATCC 12,488) is inoculated from a slant into 250ml. shake flasks containing 50 ml. of the following medium: 2% Edamin(Sheffield Farms), 5% glucose, and 0.5% corn steep liquor. After a 24hour incubation at 28 C., 10 mg. of6e-fluoro-17a-chloroethynyl-4-androstene-i75-methoxy-3-one is added toeach shake flask as a dimethyiformamide solution (100 mg./ ml.). After aconversion period of 24 hours, the cells are removed by filtration,followed by three successive extractions with equal volume of ethylacetate. The extracts are combined and concentrated in vacuo.

The 6a-fluoro-17a-chloroethynyl-1,4-androstadiene-17B- methoxy-3-one isreadily crystallized from the concentrate. Descending paperchromatography of the product in a system using formarnide as thestationary phase and chloroform as the mobile phase, shows some of thesteroid substrate, but is largely the somewhat more polar A'-dehydrogenation product.

In accordance with the above procedures, but using the 6x-fluoro-17e-bromoethynyl-4-androstene-17B methoxy- S-one in place ofthe Git-fluoro-17a-chloroethynyl-4-androstene-17/3-methoxy-3-one, thereis obtained the6oc-filloro-lh-brornoethynyl-1,4-androstadiene-l7fi-methoxy 3- one.

Example 28 A suspension of 1 g. of Goa-ilUOIO-l7cc-Cl'1l0r06tllYHYl-4-androstene-17,B-methoxy 3-one, 2.4 g. chloranil, 30 ml. ethyl acetateand 6 ml. of acetic acid is stirred and reif; .1 fluxed for 16 hours.The reaction mixture is cooled and filtered. The product is Washedsequentially with aqueous bisulr'ite, aqueous potassium hydroxide andwater. The organic layer is dried and concentrated in vacuo.Chromatography yields -fluoro-l7a-chloroethynyl 4,6-androstadiene-l75-methoxy-3-one.

Example 29 To 120 mg. ofS-iiuoro-l7cochloroethynyl-4,6-androstadiene-l7fi-methoxy-3-one in ml.of t-butanol and 0.1 ml. of acetic acid is added 50 mg. of solo umdioxide. The mixture is refluxed under nitrogen for l8 hours; then 50mg. of selenium dioxide is added and the mixture is refluxed anadditional 24- hours. The product is filtered, and the filtrateevaporated to dryness. The residue is extracted with ethyl acetate andthe extract is washed successively with aqueous sodium bicarbonate,water, dilute hydrochloric acid and Water, and then dried over magnesiumsulfate. The extract is treated with activated charcoal and thenconcentrated to dryness. Crystallization of the residue givesfi-fiuoro-l7u-chloroethynyl-1,6- androstatriene-l7fl-methoxy-3-one.

Scpzomyxa afifinis (ATCC 6,737) is inoculated from a slant into 250 ml.xe sks containing 50 ml. of the following medium: 2% Edamin (ShefieldFarms), 5% glucose, and 0.5% corn steep liquor. fter a 48 hourincubation at 28 C., of6lluoro-l7a-chloroethynyl-4,6-androstadiene-l7e-methoxy-3one is added toeach shake flask as a dimethylfo-r nanide solution (100 rug/ml). After aconversion period of 24 hours, the cells are removed by filtration,followed by three successive extractions with equal volume of ethylacetate. The extracts are combined and concentrated in vacuo.

The 6-fll101O-l7u-Chi roethynyl 1,4,6 androsta-triene-17,8-rnethoxy-3-one is readily crystallized from the concentrate.Descending paper chromatography of the product in a system usingiormamide as no stationary phase and chloroform as the mobile 12183,shows some of the steroid substrate, but is largely the somewhat morepolar A'- dehydrogenation product.

Bacillus splmericus (ATC 12,488) is inoculated from a slant into 250 ml.shake fiaslcs containing 50 m1. of the following medium: 2% Edamin(Sheffield Farms), 5% glucose, and 0.5% corn steep liquor. After a 24hour incubation at 28 C., 10 mg. of6-fiuoro-l7a-chloroethynyl-4,6-androstadiene-l7fi-rnethoxy-3-one isadded to each shake fiasx as a dimethylformarnide solution (100mg./rnl.). Afte a conversion period or 24 hours, the cells are removedby filtration followed by three successive extractions with equal volumeof eth l acetate. The extracts are combined and concentrated in vacuo.

The G-fiuoro l7ct-chloroethynyl-1,4,6-androstatriene- 17B'methoxy-3-oneis readily crystallized from the concentrate. Descending paperchromatography of the product in a system using formamide as thestationary phase and chloroform as the mobile phase, shows some o-fthesteroid substrate, but is largely the somewhat more polar ddehydrogenation product.

in accordance with the above procedure, but using the a-flllOl'Ol7el-bromoethynyl-4,o-androstadiene-l7B-methoxy-3-one in place of the6ol-iluoro-l7a-chloroethynyl- 4,6-androstadiene-l7f3-methoxyr3-one,there is obtained the -fiuoro-lh-bromoet ynyl-l,4,6 androstatriene-UB-metho-xy-B-one.

Example 31 A mixture of 1 g. of l7a-chloroethynyl-l9-nor-4-anrostenel7fl-ol-3-one, 10 ml. of acetic anhydride and 100 mg. ofp-toluenesulfonic acid is heated on the steam bath for one hour andallowed to stand overnight at room temperature. It is then poured intoice Water and left to stand at room temperature for one half hour. Thereaction mixture is then extracted with ether. The ether extract iswashed with water and aqueous sodium bicarbonate, dried and concentratedto yield the crude 17o:-

chlorcethynyl-l9 nor 3,5 androstadiene 3,l7[3,diol

diacetate. I

A solution of 8 gms. of l7a-chloroethynyl-l9-nor-3,5-androstadiene-B,l7fl-diol diacetate in a mixture of 700 m1. of ethanoland 300 ml. of tetrahydrofu ran is cooled to 10 C. and added dropwise,with occasional stirring, over a one hour period, to a cold (0 C.)solution of 18 g. sodium borohydride in 400 ml. of 80% ethanol, thereaction temperature not being allowed to exceed 5 C. After completionor the addition, the solution is held at 0-5 C. for an additional twohours. A solution of NaH PO is then added to adjust the pH to about 5.The mixture is then concentrated in vacuo to a small volume, dilutedwith Water and extracted with chloroform. The extract is Washed withwater, dried and concentrated. The concentrate is chromatographed overacid-washed alumina and eluted With ether-petroleum ether mixtures togive I7OL-Cl'llOZ'OCthYI1Yl-19 nor S-androstene 35,175 diol l7-acetate.

Ten grams of l7cl-chloroethynyl-l9-nor-5-androstene- 38,l7,8-diol-l7-acetaite in 150 ml. of tetrahydrofuran is treated withml. of 0.9 m. monoperphthalic acid in ethyl acetate and allowed to standover night at room temperature. The reaction mixture is diluted withethyl acetate and Washed sequentially with aqueous sodium bicarbonate,sodium bisulfi te, and sodium bicarbonate solution. The organic layer isdried and concentrated in vacuo. product is fractionflly crystallizedfrom aqueous ethanol to yield thel7e-chloroethynyl-l9-nor-androstane-35J76- diol-5,6a-oxide-l7-acetate.

Five grams of 17a-chlor0ethynyl-l9-nor-androstane-35,l7,8-diol-5,6a-oxide-l7-acetate in benzene is added to a reagentprepared from 3.4 g. of magnesium, 10 ml. of methyl iodide and 45 ml. ofether.

and 70 ml. of solvent is removed by distillation. After refluxing for 5hours, the mixture is cooled, acidifiedwith dilute hydrochloric acid andthe organic layer" The organic dried and Crystallization from aqueousethanol gives the 6,8-rnethyl-17a-c lorocth nyl-l enon;

Washed to neutrality. concentrated in vacuo.

give 6,8 methyl-l7a-chlorocthynyl-lQ-nor-androstan 17,8-diol-3-one.

The mixture is stirred in 2.7 g. of;

'th 1 The ether extract? is Washed sequentially with water, and aqueoussodium bicarbonate and then dried and concentrated in vacuo to The crudeproduct, dissolved in nil.

of methanol, 75 ml. or" 1 N sodium hydroxide is added and the mixture isstirred at room temperature under nitrogen for 24 hours. The mixture isconcentrated to half volume under vacuo at 25 C. and then poured intoice water. The product is extracted with ether. The ether layer isWashed with water, dried and concentrated. The concentrate ischromatographed on acid-washed alumina and the product eluted withether-petroleum ether mixtures to yield6a-methyl17oc-chloroethynyl-19-nor-4- androstene-17B-ol-3-one.

In accordance with the above procedures, but starting with the17a-bromoethynyl-19-nor-4-androstene-175-01-3- one in place of the17a-chl0roethynyl-l9-nor-4-androstene-l7 8-ol-3-one there is obtained asintermediates, the 17a-bromoethynyl-19-nor-3,5-androstadiene-3,17,B-dioldiacetate, 17a-bromoethynyl-19-nor-5-androstene-3fl,175- diol,17a-bromoethynyl-19-nor-androstane-3B,17,8-diol-5, 6m-oxide-17-acetate,and 6fl-methyl-17e-bromoethynyl-19- nor-androstane-ilfifia,17/3-trio1,and as product, the 6&- methyl 17a bromoethyny-l 19 nor 4 androstene-17fl-ol-3-one.

Example 32 100 mg. of6a-methyl-17a-chloroethynyl-19-nor-4-androstene-17fi-ol-3-one is heatedwith 1 cc. of acetic anhydride and 1.2 cc. of pyridine on the steam bathovernight. The reaction mixture is then poured onto ice and extractedwith chloroform. The extract is washed with water and concentrated. Theconcentrate is chromatographed over acid-Washed alumina and eluted withmixtures of ether-petroleum and ether to give6a-methyl-l7achloroethynyl-19-nor-4-androstene-17,6-ol-3-one acetate.

In accordance with the above procedures, but starting with theGet-methyl 17a bromoethynyl-l9-nor-4-androstene-l7,B-ol3-oue in place ofthe 6a-methyl-17u-chloroethynyl-l9-nor-4-androstene-17B-0l-3-0ne, thereis obtained the6d-methyl-17wbromoethynyl-19-nor-4-androstene-l7fi-ol-3-one acetate.

A mixture of (3-0 mg. of the6m-methyl-17a-chloroethynyl-l9-nor-4-androstene-17,8-ol-3-one, 10 ml. ofdimethylformamide, ml. of methyl iodide, and 1.5 grns. of silver oxideis stirred at room temperature for 4 days. An additional /2 gm. ofsilver oxide is added at the end of each day. One hundred ml. ofchloroform is then added to the reaction mixture which is then stirredfor one hour, filtered, and the filtrate is evaporated to dryness. Theresidual oil is chromatographed with acid- Washed alumina and elutedwith mixtures of ether and petroleum ether to give6wmethyl-17u-chloroethynyl-19- nor-4-androstene-17,8-methoxy-3-one.

In accordance with the above procedures, but starting with the Ga-methyl17a hromoethynyl-l9-nor-4-androstene-17B-ol-3-one in place of the6ca-I1lfiithYl-l7ot-Chl0f0- ethynyl-19-nor-4-androstene-176-0l-3-one,there is obtained the6ix-methyl-17a-bromoethynyl-l9-nor-4-androstene-17,6-metleoxy-3-one.

Example 33 To a solution of 200 mg. of the 17a-chloroethynyl-1-nor-3,5-androstadier1e-3,17,8-diol diacetat (Example 17) dissolved in 2ml. of acetic acid is added, with stirring, 56 ml. ofN-chlorosuccinirnide and 2 ml. of tetrahydrofuran containing 5% of dryHCl. The reaction is stirred at room temperature for 2 /2 hours. Sodiumbicarbonate is added. The reaction mixture is then extracted With ether.The ether extracts are dried and concentrated. The material ischromatographed over acidwashed alumina, and eluted with ether-petroleumether mixtures to 7 give 6u-chloro-i7a-chloroethynyLl9nor-androstene-l'ifi-ol-S-one acetate.

in accordance with the above procedures, but starting with thel71xbromoethynyl-19-nor-3,5-audrcstadieue-3, 17,6-diol diacetate inplace of the 17a-chloroethynyl-19- nor-",S-androstadiene 3,175 dioldiacetate there is obtained the 6m chloro 17abromoethynyl-l9-nor-androstene-17,B-ol-3-one acetate.

3% Example 34 A solution or" the17a-chloroethynyl-l9-nor-3,S-androstadiene-3,17fl diol diacetate inaqueous tetrahydrofuran is treated with a slow stream of perchlorylfluoride for one hour and then allowed to stand at room temperature foran additional 2 hours. The reaction mixture is poured on ice andextracted with chloroform. The chloroform layer is washed with aqueousbicarbonate solution, dried and concentrated in vacuo. The crudematerial is dissolved in acetic acid saturated with gaseous hydrogenchloride and allowed to stand for one hour at room temperature. Thereaction mixture is poured on ice and extracted with chloroform. Thechloroform layer is Washed with aqueous bicarbonate solution, dried andconcentrated in vacuo. The concentrate is chromatographed on acid-washedalumina to yield the 6a-fluoro-17u-chloroethynyl-19-nor-androstene-17B-ol 3 one acetale.

in accordance with the above procedures, but starting with the 17::-bromoethynyl-19-nor-3,5-androstadiene-3, 17fl-diol diacetate in placeof the 17a-chloroethynyld9- nor-3,5-androstadiene 3,1713 diol diacetatethere is obtained the 6st-fluoro-17e-bromoethynyl-19-nor-4-androstene-17 3-o1-3-one acetate.

A mixture of 500 mg. of the 17a-chloroethynyl-19-nor- 4-androstene-17,8ol-3-one (Example 3) 10 m1. of dirnethylformamide, 20 ml. of methyliodide, and 1.5 gm. of silver oxide is stirred at room temperature for 4days. An additional A2 gm. of silver oxide is added at the end of eachday. One hundred ml. of chloroform is then added to the reaction mixturewhich is then stirred for one hour, filtered, and the filtrate isevaporated to dryness. The residual oil is chromatographed withacid-washed alumina and eluted with mixtures of ether and petroleumether to give 17a-chloroethynyl-19-nor-4-androstene-17B- methoxy-3-one.

In accordance with the above procedurm, but starting with the17a-bromoethynyl-19-nor-4 androstene-175-01- 3-one in place of the17a-chloroethynyl-19-nor-4-androstene-17fi-ol-3-one there is obtainedthe 17a-bromoethynyl-19-nor-4-androstone-17(3-methoxy-3-or1e.

A mixture of 1 g. of l'la-ClflOI'OtithYHYl-l9-I1OI-4-fi11d10-stene-17B-methoxy-3-one, 10 ml. of acetic anhydride and mg. ofp-toluenesulfonic acid is heated on .the steam bath for one hour andallowed to stand overnight at room temperature. It is then poured intoice water and left to stand at room temperature for one half hour. Thereaction mixture :is then extracted with ethe The ether extract iswashed with Water and aqueous sodium bicarbonate, dried, andconcentrated to yield the crude 17achioroethynyl 19 nor 3,5androstadiene 17B- methoxy-B-ol acetate.

In accordance with the above procedures, but starting with the17tz-brornoethynyl-19-nor-4-andnostene-175-mothoxy-Z-one in place of the17a-ch1oroethynyl-19--nor- 4-androstene-17/3-methoxy-3-one, there isobtained the 17cc romoethy-nyl 19 nor 3,5 androstadiene 17erneth0xy-3olacetate.

To a solution of 200 mg. of the 17a-chloroethynyl-19nor-3,S-androstadiene-l7/3-methox* -3-ol acetate dissolved in 2 ml. ofacetic aoid is added, with stirring, 56 m1. of N-chlorosucoinimide and 2ml. of tetrahydrofuran contaming 5% of dry HCl. The reaction is stirredat room temperature for 2 /2 hours. Sodium bicarbonate is added. Thereaction mixture is then extracted With ether. The ether extracts aredried and concentrated. The material is chromatographed over acid-washedalumina, and eluted with ether-petroleum ether mixtures to give6u-Chl010- 17oz ohloroethynyl 19 nor 4 androstene 17/3- methoxy-3-one.

In accordance with the above procedures, but starting with the17a-bromoethynyh19-nor-3,5-androstadiene-17B- methoxy-3-ol acetate :inplace of the 17a-chlo-roeflhynyl-19-nor-3,5-androstadiene-l7p-methoxy-3eol acetate there is obtained theoat-chloro-l7a-brornoethynyl-l9-nor-4- androstene-l7B-methoxy-3-one.

Example 35 A solution of thel7a-chloroethynyl-l9-nor-3,5-androstadiene-l7fi-methoxy-3wl acetate inaqueous tetrahydrofuran is treated with a slow stream of perchlorylfluoride for one hour and then allowed to stand at room temperature foran additional 2 hours. The reaction mixture is poured on ice andextracted with chloroform. The chloreform layer is Washed with aqueousbicarbonate solution, dried and concentrated in vacuo. The crudematerial is dissolved in acetic acid saturated With gaseous hydrogenchloride and allowed to stand for one hour at room temperature. Thereaction mixture is poured on ice and extracted with chloroform. Thechloroform layer is washed with aqueous bicarbonate solution, dried andconcentrated in vacuo. The concentrate is chromatographed on acid-Washedalumina to yield the 6e-fluoro- 170; chloroethynyl 19 no-r 4 androstene17,8- methoxy-3-one.

in accordance With the above procedures, but starting with thel7a-brornoetliynyl-19-nor-3,5'-androstad=iene-l7 methoxy-3-ol acetate inplace of the l7ot-chloroethynyl-19-nor-3,S-anclrostadiene-17B-methoxy-3-ol acetate, there is obtainedthe 5cc-fill0l0-I'YuebIOmOBlZhYHYl-19-I10I-4- androstenel7/3-methoxy-3-one.

Example 36 A solution of 100 mg. of the l7a-chloroethynyl-l9-ncr-4-andros-tene'17e-ol-3-one and 50 mg. of Lindlar catalyst in 10 cc. ofethyl acetate is treated with hydrogen until one mole of hydrogen hasbeen absorbed. The mixture is filtered and concentrated to yield thecrude chlorovinyl compound. Chromatography yields the pure product 21-chloro-l9-nor-4,20-pregnadiene-l7fi-ol-3-one.

in accordance with the above procedure, but starting with the17ot-bromoethynyl-19-nor-4-androetene-175-01-3- one, thel7u-chloroethynyl-19-nor-5 l) -androstene-l7fiol-3-one, or thel7a-brornoethynyl-l9-nor-5(l0)-androstene-175-ol-3-one, in place of the17a-chl r0ethynyl-19- nor-4-androstene-l7B-ol3-one, there is obtainedthe 21- bromo-19-nor-4,20-preg1adiene-l7,8-ol-3-one, ZI-chloro- 19-nor-5l0) ,29-pregnadiene-17B-ol-3 -one, or 21-bromo- 19-nor-5 l0),ZG-pregnadiene-l'7B-ol-3-one respectively.

Example 37 A suspension or" platinum oxide in 10 cc. of ethanol iseduced and 100 mg. of 17a-chloroethynyl-l9-nor-4-androstene-l7B-ol-3-one is added. Reduction proceeds until two moles ofhydrogen have been absorbed. The solution is filtered, concentrated andehrornatographed on alumina to yield the2l-chloro-l9-nor-4-preg1ene-l7flol- 3-one.

In accordance with the above procedure, but starting with the17u-brornoethynyl-l9-nor4-androstene-17,8-ol-3- one, thel7achloroethynyl-l9-nor-5 l 0) -androstene-17B- ol-3-one, or the17oc-b1'OrnOSthYl1Yl-l9-110r-5 (10)4J1dl'0 stone-17,8-ol-3-one in placeof the 17a-chloroethynyl-l9- nor4-androstene-l7B-ol-3-one, there isobtained the 21- bromol9nor-4pregnene-l75-ol-3-one, the 2l-chloro-l9-nor-(10)pregnenel7B-ol-3-one, or the 2l-bromo-l9 nor-5 lO).-regnene-l7fi-ol-3-one respectively.

claim:

1. 17a bromoethynyl-l9-nor-5 lO)-androstene-l7fi-ol- 3one acetate. 7

2. 17oz oromoethynyl l9-nor-5(lO)-androstene-l7fi methoxy-3-one.

3. Process for preparation of a mixture of A and A-17wethynyl-3ethylenedioxy l9 nor-androstenel7f3-ols which comprisesreacting l7a-ethynyl-l9-nor-4- androstene-l7B-ol-3-one with ethyleneglycol.

4. Process for the preparation of a mixture of A and M -17a ethynyl3-ethylenedioxy-l9-nor-androstene- 17,6-01 tetrahydropyranyl etherswhich comprises reacting a mixture of A and A-l7zx-ethynyl-3ethylenedioxy- 19-nor-androstene-l7fi-ols withdihydropyran in the pres ence of an acidic reagent.

5. Process for the preparation of a mixture of the A and A-l7u-haloethynyl-3-ethylenedioxyl9-nor androstene-17p-oltetrahydropyranyl ethers which comprises re; acting a mixture of the Aand A -l7u-ethynyl-3-ethylenedioxy-l9-nor-androstene47,8 oltetrahydropyranyl ethers with a halogenating agent.

6. Process for the preparation of a mixture of the A and AUCi-chIOroethynyl 3 ethylenedioxy-l9-norandros.ene-1'ifi-ol-tetrahydropyra-nyl others which corn- ;rises T536115a mixture of the A and M -'17- ethynyl-Z-ethylenedioxy-l9-nor-androstene17B-ol-tetrahydropyranyl ethers, dissolved in tertiary butyl alcohol,sequentially with an alkali metal tertiary butoxide, and then withtertiary butyl hypochlorite.

7. Process for the preparation of a mixture of the A and A 17abromoethynyl-3-ethylenedioxy-l9-nor androstene-l7,8-ol-tetrahydropyranylothers which compris reacting a mixture of the A and A -l7a-ethynyl#3-ethylenedioxy-l9 nor-androstene 17,e-ol-tetrahydro pyranyl ethers,dissolved in tertiary butyl alcohol, .se-

9. 170a ethyny 3-ethylenedioxy-l9-nor-5-androstcne- 17,6-01tetrahydropyranyl ether. 7

10. 17a chloroethynyl 3-ethylenedioxy-l9-nor-5- androstene-lm-oltetrahydropyranyl ether.

11. 17a chloroethynyl-3-ethylenedioxy-l9-nor-5(l0)- androstene-Ufi-oltetrahydropyranyl ether.

androstene-Ufil tetrahydropyranyl ether.

13. 17st bromoethynyl-B-ethyienedioxy-l9-nor-5(l0)-. androstene-l'Zfi-oltetrahydropyranyl ether.

14. A compound selected from the group consisting of17ebromoethynyl-l9-nor-5(l0)-androstene 17,8-01-3 one, and the17,6-lower allianoyl esters and the fill-lower alkyl ethers thereof.

No references cited.

12. 17oz bromoethynyl 3 ethyienedioxy-lQ-nor-S-

3. PROCESS FOR THE PREPARATION OF A MIXTURE OF A5 ANDA5(10)-17A-ETHYNYL-3-ETHYLENEDIOXY-19-NOR-ANDROSTENE17B-OLS WHICHCOMPRISES REACTING 17A-ETHYNYL-19-NOR-4ANDROSTENE-17B-OL-3-ONE WITHETHYLENE GLYCOL. 10.17A-CHLOROETHYNYL-3-ETHYLENEDIOXY-19-NOR-5ANDROSTENE-17B-OLTETRAHYDROPYRANYL ETHER.